Non-human animals expressing humanized c1q complex

ABSTRACT

Disclosed herein are nucleic acids encoding for and proteins expressing chimeric C1q polypeptides, non-human animals comprising said nucleic acids, and methods of making or using said non-human animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/144,156, filed Sep. 27, 2018, which claims the benefit of priorityfrom U.S. Provisional Application No. 62/565,438, filed Sep. 29, 2017,the contents of which are incorporated herein by reference in itsentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as35342Z_10353US02_SequenceListing.txt of 50 KB, created on Oct. 20, 2021and submitted to the United States Patent and Trademark Office viaEFS-Web, is incorporated herein by reference.

BACKGROUND

During preclinical drug development stage, candidate agents aretypically studied based on their efficacy, toxicity, and otherpharmacokinetic and pharmacodynamics properties. Candidate agents, suchas antibodies, typically target a human antigen—as the end goal ofinvestigation is to develop a human therapy. The ability to sequesterthe complement pathway provides a significant advantage to candidatetherapeutic agents. The complement pathway is part of the innate immuneresponse and assists humoral immune responses in the recruitment ofmarcrophage and phagocytes to the antigenic site. Activation of thecomplement pathway results in cytokine release and the opsonization ofthe antibody-bound antigen by phagocytes. During development oftherapeutic agents that are aimed at activation of complement pathwayand innate immune response in order to combat human disease, a modelnon-human animal system that would allow studies into the mechanisms ofaction and/or therapeutic potential of the agent is invaluable; but suchsystem is lacking.

SUMMARY

Disclosed herein are chimeric mammalian C1q polypeptides (such as, forexample chimeric mammalian C1qa, C1qb and/or C1qc polypeptides), nucleicacid molecules encoding chimeric mammalian C1q polypeptides, andnon-human animals (e.g., mammals such as rodents) comprising saidnucleic acid molecules and expressing chimeric C1q polypeptides.

In one aspect, disclosed herein is a genetically modified non-humananimal comprising in its genome a nucleic acid encoding a chimeric C1qpolypeptide (e.g., a chimeric C1qa polypeptide, a chimeric C1qbpolypeptide, or a chimeric C1qc polypeptide), wherein the nucleic acidcomprises a non-human nucleic acid sequence and a human nucleic acidsequence.

In some embodiments, the genetically modified non-human animal comprisesin its genome more than one nucleic acid encoding a chimeric C1qpolypeptide; for example, the non-human animal comprises in its genome acombination (e.g., two or all three) of a nucleic acid encoding achimeric C1qa polypeptide, a nucleic acid encoding a chimeric C1qbpolypeptide, and a nucleic acid encoding a chimeric C1qc polypeptide.

In some embodiments, the non-human animal is a mammal. In someembodiments, the non-human animal is a rodent, such as a rat or a mouse.

In some embodiments, the chimeric C1q polypeptide comprises a globularhead domain that is substantially human (i.e., substantially identicalto the globular head domain of a human C1q polypeptide), and anN-terminal stalk-stem region that is substantially non-human (i.e.,substantially identical to the N-terminal stalk-stem region of anon-human C1q polypeptide such as an endogenous C1q polypeptide).

In some embodiments, the non-human animal comprises a nucleic acidencoding a chimeric C1q polypeptide that is a chimeric C1qa polypeptide.In some embodiments, the chimeric C1qa polypeptide comprises a globularhead domain that is substantially identical to the globular head domainof a human C1qa polypeptide, and an N-terminal stalk-stem region that issubstantially identical to the N-terminal stalk-stem region of anon-human C1qa polypeptide such as an endogenous C1qa polypeptide. Insome embodiments, the globular head domain of a human C1qa polypeptidecomprises amino acids 108-245 of SEQ ID NO: 4.

In some embodiments, the non-human animal is a mouse which comprises anucleic acid encoding a chimeric C1qa polypeptide that comprises aglobular head domain that is substantially identical to the globularhead domain of a human C1qa polypeptide, and an N-terminal stalk-stemregion that is substantially identical to the N-terminal stalk-stemregion of a mouse C1qa polypeptide such as an endogenous mouse C1qapolypeptide. In certain embodiments, the N-terminal stalk-stem region ofthe endogenous mouse C1qa polypeptide comprises amino acids 23-107 ofSEQ ID NO: 1. In specific embodiments, the chimeric C1qa polypeptidecomprises amino acids 23-245 of SEQ ID NO: 10 (mouse/human). In specificembodiments, the chimeric C1qa polypeptide comprises the amino acidsequence of SEQ ID NO: 10 (mouse/human).

In some embodiments, the genetically modified non-human animal is a rat,which comprises a nucleic acid encoding a chimeric C1qa polypeptide thatcomprises a globular head domain that is substantially identical to theglobular head domain of a human C1qa polypeptide, and an N-terminalstalk-stem region that is substantially identical to the N-terminalstalk-stem region of a rat C1qa polypeptide such as an endogenous ratC1qa polypeptide. In some embodiments, the N-terminal stalk-stem regionof the endogenous rat C1qa polypeptide comprises amino acids 23-107 ofSEQ ID NO: 7. In specific embodiments, the chimeric C1qa polypeptidecomprises amino acids 23-245 of SEQ ID NO: 55 (rat/human). In specificembodiments, the chimeric C1qa polypeptide comprises the amino acidsequence of SEQ ID NO: 55 (rat/human).

In some embodiments, the non-human animal comprises a nucleic acidencoding a chimeric C1q polypeptide that is a chimeric C1qb polypeptide.In some embodiments, the chimeric C1qb polypeptide comprises a globularhead domain that is substantially identical to the globular head domainof a human C1qb polypeptide, and an N-terminal stalk-stem region that issubstantially identical to the N-terminal stalk-stem region of anon-human C1qb polypeptide such as an endogenous C1qb polypeptide. Insome embodiments, the globular head domain of a human C1qb polypeptidecomprises amino acids 115-251 of SEQ ID NO: 5.

In some embodiments, the non-human animal is a mouse, which comprises anucleic acid encoding a chimeric C1qb polypeptide that comprises anN-terminal stalk-stem region that is substantially identical to theN-terminal stalk-stem region of a mouse C1qb polypeptide such as anendogenous mouse C1qb polypeptide. In some embodiments, the N-terminalstalk-stem region of the endogenous mouse C1qb polypeptide comprisesamino acids 26-114 of SEQ ID NO: 2. In specific embodiments, thechimeric C1qb polypeptide comprises amino acids 26-251 of SEQ ID NO: 11(mouse/human). In specific embodiments, the chimeric C1qb polypeptidecomprises the amino acid sequence of SEQ ID NO: 11 (mouse/human).

In some embodiments, the non-human animal is a rat, which comprises anucleic acid encoding a chimeric C1qb polypeptide that comprises anN-terminal stalk-stem region that is substantially identical to theN-terminal stalk-stem region of a rat C1qb polypeptide such as anendogenous rat C1qb polypeptide. In some embodiments, the N-terminalstalk-stem region of the endogenous rat C1qb polypeptide comprises aminoacids 26-114 of SEQ ID NO: 8. In specific embodiments, the chimeric C1qbpolypeptide comprises amino acids 26-251 of SEQ ID NO: 56 (rat/human).In specific embodiments, the chimeric C1qb polypeptide comprises theamino acid sequence of SEQ ID NO: 56 (rat/human).

In some embodiments, the non-human animal comprises a nucleic acidencoding a chimeric C1q polypeptide that is a chimeric C1qc polypeptide.In some embodiments, the chimeric C1qc polypeptide comprises a globularhead domain that is substantially identical to the globular head domainof a human C1qc polypeptide, and an N-terminal stalk-stem region that issubstantially identical to the N-terminal stalk-stem region of anon-human C1qc polypeptide such as an endogenous C1qc polypeptide. Insome embodiments, the globular head domain of a human C1qc polypeptidecomprises 113-245 of SEQ ID NO: 6.

In some embodiments, the non-human animal is a mouse which comprises anucleic acid encoding a chimeric C1qc polypeptide that comprises aglobular head domain that is substantially identical to the globularhead domain of a human C1qc polypeptide, and an N-terminal stalk-stemregion that is substantially identical to the N-terminal stalk-stemregion of a mouse C1qc polypeptide such as an endogenous mouse C1qcpolypeptide. In certain embodiments, the N-terminal stalk-stem region ofthe endogenous mouse C1qc polypeptide comprises amino acids 30-113 ofSEQ ID NO: 3. In specific embodiments, the chimeric C1qc polypeptidecomprises amino acids 30-246 of SEQ ID NO: 12 (mouse/human). In specificembodiments, the chimeric C1qc polypeptide comprises the amino acidsequence of SEQ ID NO: 12 (mouse/human).

In some embodiments, the genetically modified non-human animal is a rat,which comprises a nucleic acid encoding a chimeric C1qc polypeptide thatcomprises a globular head domain that is substantially identical to theglobular head domain of a human C1qc polypeptide, and an N-terminalstalk-stem region that is substantially identical to the N-terminalstalk-stem region of a rat C1qc polypeptide such as an endogenous ratC1qc polypeptide. In some embodiments, the N-terminal stalk-stem regionof the endogenous rat C1qc polypeptide comprises amino acids 32-115 ofSEQ ID NO: 9. In specific embodiments, the chimeric C1qc polypeptidecomprises amino acids 32-248 of SEQ ID NO: 57 (rat/human). In specificembodiments, the chimeric C1qc polypeptide comprises the amino acidsequence of SEQ ID NO: 57 (rat/human).

In some embodiments, a chimeric C1q polypeptide is translated in thenon-human animal to contain a non-human C1q signal peptide such as anendogenous non-human C1q signal peptide. In other words, the nucleicacid molecule encoding a chimeric C1q polypeptide also comprises acoding sequence for a non-human C1q signal peptide such as an endogenousC1q signal peptide. For example, a nucleic acid molecule encoding achimeric C1qa polypeptide also comprises a coding sequence for anon-human C1qa signal peptide such as an endogenous C1qa signal peptide;a nucleic acid molecule encoding a chimeric C1qb polypeptide alsocomprises a coding sequence for a non-human C1qb signal peptide such asan endogenous C1qb signal peptide; and a nucleic acid molecule encodinga chimeric C1qc polypeptide also comprises a coding sequence for anon-human C1qc signal peptide such as an endogenous C1qc signal peptide.Examples of mouse and rat C1q signal peptides are disclosed herein (see,e.g., in FIGS. 3A-3C).

In some embodiments, the nucleic acid encoding a chimeric C1qpolypeptide is at a locus other than an endogenous non-human C1q locus.In other embodiments, the nucleic acid encoding a chimeric C1qpolypeptide is at an endogenous non-human C1q locus. For example, anucleic acid encoding a chimeric C1qa polypeptide is at an endogenousnon-human C1qa locus; a nucleic acid encoding a chimeric C1qbpolypeptide is at an endogenous non-human C1qb locus; and/or a nucleicacid encoding a chimeric C1qc polypeptide is at an endogenous non-humanC1qc locus.

In embodiments where the nucleic acid encoding a chimeric C1qpolypeptide is at an endogenous non-human C1q locus, in some suchembodiments, an endogenous genomic sequence at the endogenous non-humanC1q locus has been replaced by a human nucleic acid sequence. In someembodiments, the human nucleic acid sequence, such as a genomic fragmentof a human C1q gene, encodes substantially the globular head domain of ahuman C1q polypeptide. In some embodiments, the human nucleic acidsequence also includes the 3′ UTR of the human C1q gene (including thepolyadenylation signal and the polyadenylation site of the human C1qgene).

In some embodiments, a genetically modified non-human animal comprises anucleic acid encoding a chimeric C1qa polypeptide, wherein the nucleicacid comprises human and non-human nucleic acid sequences, and whereinthe human nucleic acid sequence encodes substantially the globular headdomain of a human C1qa polypeptide. In some embodiments, the globularhead domain of the human C1qa polypeptide comprises amino acids 108-245of SEQ ID NO: 4. In some embodiments, the human nucleic acid sequenceencodes amino acids 112-245 of SEQ ID NO: 4.

In some embodiments, a genetically modified non-human animal comprises anucleic acid encoding a chimeric C1qb polypeptide, wherein the nucleicacid comprises human and non-human nucleic acid sequences, and whereinthe human nucleic acid sequence encodes substantially the globular headdomain of a human C1qb polypeptide. In some embodiments, the globularhead domain of the human C1qb polypeptide comprises amino acids 115-251of SEQ ID NO: 5. In some embodiments, the human nucleic acid sequenceencodes amino acids 118-251 of SEQ ID NO: 5.

In some embodiments, a genetically modified non-human animal comprises anucleic acid encoding a chimeric C1qc polypeptide, wherein the nucleicacid comprises human and non-human nucleic acid sequences, and whereinthe human nucleic acid sequence encodes substantially the globular headdomain of a human C1qc polypeptide. In some embodiments, the globularhead domain of the human C1qc polypeptide comprises amino acids 113-245of SEQ ID NO: 6. In some embodiments, the human nucleic acid sequenceencodes amino acids 114-245 of SEQ ID NO: 6.

In embodiments of a genetically modified non-human animal comprising anucleic acid encoding a chimeric C1q polypeptide, wherein the nucleicacid comprises human and non-human nucleic acid sequences, the non-humannucleic acid sequences encode substantially the N-terminal stalk-stemregion of a non-human C1q polypeptide such as an endogenous non-humanC1q polypeptide. In embodiments where an endogenous genomic sequence atthe endogenous non-human C1q locus has been replaced by a human nucleicacid sequence, in some such embodiments, the endogenous genomic sequenceremaining at the C1q locus encodes substantially the N-terminalstalk-stem region of the endogenous C1q polypeptide.

In some embodiments, the non-human animal is a mouse, and the N-terminalstalk-stem region of an endogenous C1q polypeptide comprises amino acids23-107 of SEQ ID NO: 1 (for C1qa), amino acids 26-114 of SEQ ID NO: 2(for C1qb), or amino acids 30-113 of SEQ ID NO: 3 (for C1qc). In someembodiments, the mouse comprises a nucleic acid encoding a chimeric C1qapolypeptide, wherein the nucleic acid comprises a mouse nucleic acidsequence and a human nucleic acid sequence, and wherein the mousenucleic acid sequence encodes amino acids 23-111 of SEQ ID NO: 1. Insome embodiments, the mouse comprises a nucleic acid encoding a chimericC1qb polypeptide, wherein the nucleic acid comprises a mouse nucleicacid sequence and a human nucleic acid sequence, and wherein the mousenucleic acid sequence encodes amino acids 26-117 of SEQ ID NO: 2. Insome embodiments, the mouse comprises a nucleic acid encoding a chimericC1qc polypeptide, wherein the nucleic acid comprises a mouse nucleicacid sequence and a human nucleic acid sequence, wherein the mousenucleic acid sequence encodes amino acids 30-114 of SEQ ID NO: 3.

In some embodiments, the non-human animal is a rat, and the N-terminalstalk-stem region of the endogenous C1q polypeptides comprises aminoacids 23-107 of SEQ ID NO: 7 (for C1qa), amino acids 26-114 of SEQ IDNO: 8 (for C1qb), or amino acids 32-115 of SEQ ID NO: 9 (for C1qc). Insome embodiments, the rat comprises a nucleic acid encoding a chimericC1qa polypeptide, wherein the nucleic acid comprises a rat nucleic acidsequence and a human nucleic acid sequence, wherein the rat nucleic acidsequence encodes amino acids 23-111 of SEQ ID NO: 7. In someembodiments, the rat comprises a nucleic acid encoding a chimeric C1qbpolypeptide, wherein the nucleic acid comprises a rat nucleic acidsequence and a human nucleic acid sequence, wherein the rat nucleic acidsequence encodes amino acids 26-117 of SEQ ID NO: 8. In someembodiments, the rat comprises a nucleic acid encoding a chimeric C1qcpolypeptide, wherein the nucleic acid comprises a rat nucleic acidsequence and a human nucleic acid sequence, wherein the rat nucleic acidsequence encodes amino acids 32-116 of SEQ ID NO: 9.

In a specific embodiment, the genetically modified non-human animal is arat and comprises in its genome: (i) at the endogenous C1qa locus anucleic acid sequence encoding a chimeric rat/human C1qa polypeptidewherein the nucleic acid sequence comprises, 5′-3′ and in operablelinkage a first nucleotide sequence encoding amino acids 1-111 of a ratC1qa polypeptide of SEQ ID NO: 7 and a second nucleotide sequenceencoding amino acids 112-245 of a human C1qa polypeptide of SEQ ID NO:4; (ii) at the endogenous C1qb locus a nucleic acid sequence encoding achimeric rat/human C1qb polypeptide wherein the nucleic acid sequencecomprises, 5′-3′ and in operable linkage a third nucleotide sequenceencoding amino acids 1-117 of a rat C1qb polypeptide of SEQ ID NO: 8 anda fourth nucleotide sequence encoding amino acids 118-251 of a humanC1qb polypeptide of SEQ ID NO: 5; and (iii) at the endogenous C1qc locusa nucleic acid sequence encoding a chimeric rat/human C1qc polypeptidewherein the nucleic acid sequence comprises, 5′-3′ and in operablelinkage a fifth nucleotide sequence encoding amino acids 1-116 of a ratC1qc polypeptide of SEQ ID NO: 9 and a sixth nucleotide sequenceencoding amino acids 114-245 of a human C1qc polypeptide of SEQ ID NO:6. In a particular embodiment, the genetically modified non-human animalis a rat which comprises in its genome: at the endogenous C1qa locus anucleic acid sequence encoding a chimeric rat/human C1qa polypeptidewhich comprises the amino acid sequence of SEQ ID NO: 55; at theendogenous C1qb locus a nucleic acid sequence encoding a chimericrat/human C1qb polypeptide which comprises the amino acid sequence ofSEQ ID NO: 56; and at the endogenous C1qa locus a nucleic acid sequenceencoding a chimeric rat/human C1qc polypeptide which comprises the aminoacid sequence of SEQ ID NO: 57.

In another specific embodiment, the genetically modified non-humananimal is a mouse and comprises in its genome: (i) at the endogenousC1qa locus a nucleic acid sequence encoding a chimeric mouse/human C1qapolypeptide wherein the nucleic acid sequence comprises, 5′-3′ and inoperable linkage a first nucleotide sequence encoding amino acids 1-111of a mouse C1qa polypeptide of SEQ ID NO: 1 and a second nucleotidesequence encoding amino acids 112-245 of a human C1qa polypeptide of SEQID NO: 4; (ii) at the endogenous C1qb locus a nucleic acid sequenceencoding a chimeric mouse/human C1qb polypeptide wherein the nucleicacid sequence comprises, 5′-3′ and in operable linkage a thirdnucleotide sequence encoding amino acids 1-117 of a mouse C1qbpolypeptide of SEQ ID NO: 2 and a fourth nucleotide sequence encodingamino acids 118-251 of a human C1qb polypeptide of SEQ ID NO: 5; and(iii) at the endogenous C1qc locus a nucleic acid sequence encoding achimeric mouse/human C1qc polypeptide wherein the nucleic acid sequencecomprises, 5′-3′ and in operable linkage a fifth nucleotide sequenceencoding amino acids 1-114 of a mouse C1qc polypeptide of SEQ ID NO: 3and a sixth nucleotide sequence encoding amino acids 114-245 of a humanC1qc polypeptide of SEQ ID NO: 6. In a particular embodiment, thegenetically modified non-human animal is a mouse which comprises in itsgenome: at the endogenous C1qa locus a nucleic acid sequence encoding achimeric mouse/human C1qa polypeptide which comprises the amino acidsequence of SEQ ID NO: 10; at the endogenous C1qb locus a nucleic acidsequence encoding a chimeric mouse/human C1qb polypeptide whichcomprises the amino acid sequence of SEQ ID NO: 11; and at theendogenous C1qa locus a nucleic acid sequence encoding a chimericmouse/human C1qc polypeptide which comprises the amino acid sequence ofSEQ ID NO: 12.

In some embodiments disclosed herein, the genetically modified non-humananimal does not express a functional endogenous C1qa, C1qb, and/or C1qcpolypeptide(s).

In another aspect, provided herein are methods of making a geneticallymodified non-human animal (such as, for example a non-human mammal suchas a rodent including but not limited to a mouse or rat) comprising inits genome a chimeric C1q locus that includes a gene encoding a chimericnon-human/human C1qa polypeptide, a gene encoding a chimericnon-human/human C1qb polypeptide, and/or a gene encoding a chimericnon-human/human C1qc polypeptide, the method comprising introducing intothe non-human animal genome a nucleic acid sequence(s) comprising a) agene encoding a chimeric non-human/human C1qa polypeptide, b) a geneencoding a chimeric non-human/human C1qb polypeptide, and/or c) a geneencoding a chimeric non-human/human C1qc polypeptide. In someembodiments, the nucleic acid comprising a gene encoding a chimeric C1qpolypeptide is at a location in the genome outside the endogenous locus.Thus, in some embodiments, the endogenous C1q gene(s) or a portionthereof may be silenced and/or deleted, such that the non-human animaldoes not express a functional endogenous C1q polypeptide (e.g., does notexpress a functional endogenous C1qa, C1qb, and/or C1qc polypeptide). Inother embodiments, the nucleic acid comprising a gene encoding achimeric polypeptide is at the endogenous non-human C1q locus; and inone embodiment, the nucleic acid comprising a gene encoding a chimericC1q polypeptide replaces the endogenous non-human C1q gene at theendogenous C1q locus.

Thus, provided herein are methods of making the genetically modifiednon-human animal described herein, wherein the nucleic acid sequenceencoding a chimeric C1q polypeptide is introduced at the endogenousnon-human mammal C1q locus. In a specific aspect, disclosed is a methodof making a genetically modified non-human animal, wherein the nucleicacid sequence encoding a chimeric C1q polypeptide replaces a nucleotidesequence encoding an endogenous non-human mammal C1q polypeptide.

In some embodiments, the method of making a genetically modifiednon-human animal described herein, e.g., a genetically modified rat ormouse, comprises generating a targeting vector (e.g., a large targetingvector (LTVEC)) comprising nucleic acid sequence(s) encoding a chimericC1qa, C1qb, and/or C1qc polypeptide(s), introducing said targetingvector into ES cells, and generating said non-human animal from said EScell.

In another aspect, disclosed herein is a chimeric C1q polypeptidecomprising a globular head domain that is substantially human and anN-terminal stalk-stem region that is substantially non-human, whereinthe chimeric C1q polypeptide is selected from the group consisting of achimeric C1qa polypeptide, a chimeric C1qb polypeptide, and a chimericC1qc polypeptide. In some embodiments, such a chimeric C1q polypeptideis made from the genetically modified non-human animal disclosed herein.In other embodiments, a chimeric C1q polypeptide is made from anappropriate host cell.

In still another aspect, disclosed herein is a chimeric C1q proteincomprising one or more of the chimeric C1q polypeptides disclosedherein. In some embodiments, the chimeric C1q protein comprises at leastone chimeric C1qa, one chimeric C1qb, and one chimeric C1qc polypeptide.In some embodiments, the chimeric C1q protein comprises 6 each of achimeric C1qa polypeptide, a chimeric C1qb polypeptide, and a chimericC1qc polypeptide.

In another aspect, disclosed herein is an isolated nucleic acid encodinga chimeric C1q polypeptide (a C1qa polypeptide, a C1qb polypeptide, or aC1qc polypeptide), and comprising a non-human mammal nucleic acidsequence and a human nucleic acid sequence. In some embodiments, thehuman nucleic acid sequence encodes substantially the globular headdomain of a human C1q polypeptide and the non-human nucleic acidsequence encodes substantially the N-terminal stalk-stem region of acognate non-human C1q polypeptide. In some embodiments, the isolatednucleic acid encodes a chimeric C1q protein, and comprises one or moreof a first, second or third nucleotide sequences, wherein the firstnucleotide sequence encodes a chimeric C1qa polypeptide, the secondnucleotide sequence encodes a chimeric C1qb polypeptide, and the thirdnucleotide sequence encodes a chimeric C1qc polypeptide.

In still another aspect, provided herein is a cell comprising anisolated nucleic acid disclosed herein. In some embodiments, the cell isan embryonic stem (ES) cell, e.g., a rodent (such as mouse or rat) EScell.

In a further aspect, provided herein is a rodent model for testing aC1q-based bispecific antigen-binding protein, wherein theantigen-binding protein binds both human C1q and an antigen of interest,comprising a genetically modified rodent disclosed herein and furthercomprising the antigen of interest or a cell expressing the antigen ofinterest.

In another aspect, disclosed herein are methods of screening for a drugcandidate that targets an antigen of interest comprising introducing theantigen of interest into the genetically modified non-human animal,e.g., the rodent, e.g., the rat or the mouse, provided herein,contacting said animal with a drug candidate of interest, wherein thedrug candidate is directed against the human C1q and the antigen ofinterest, and assaying to determine whether the drug candidate isefficacious in preventing, reducing, or eliminating cells characterizedby the presence or expression of the antigen of interest. In oneembodiment, the step of introducing comprises expressing in the animalthe antigen of interest (such as, for example, genetically modifying therodent that expresses the antigen of interest) or introducing into saidanimal a cell or virus expressing the antigen of interest. In onespecific aspect, the cell can be a tumor cell or bacterial cell. In afurther aspect the antigen of interest can be a tumor associated antigenor a bacterial antigen. In another aspect, the cell can be a bacterialcell.

In another aspect, provided herein is a method of screening amongtherapeutic drug candidates that target an antigen of interest, themethod comprising mixing a cell or virus expressing the antigen ofinterest with (i) a drug candidate of interest, wherein the drugcandidate is directed against the human C1q and the antigen of interest,and (ii) a blood sample (e.g., a whole blood sample) of a geneticallymodified rodent described herein, and (b) assaying to determine whetherthe drug candidate is efficacious in reducing or eliminating the cell orvirus characterized by the presence or expression of the antigen ofinterest. The determination can be made based on measuring, e.g.,percentage survival of the cell or virus where a drug candidate is usedas compared to a control drug or no drug at all. The antigen of interestmay be a tumor-associated antigen or an infectious disease associatedantigen, e.g., a bacterial or a viral antigen. In some embodiments, theantigen of interest is a bacterial antigen such as a Staphylococcusantigen. In some embodiments, the cell is a bacterial cell such as aStaphylococcus cell.

In some embodiments, provided is a method of screening for a drugcandidate, wherein the step of introducing comprises infecting theanimal (e.g., the rat or the mouse) with the antigen of interest (forexample, a viral antigen or a bacterial antigen such as a Staphylococcusantigen). Thus, in one aspect, the step of introducing comprisesinfecting the animal with a virus or bacteria.

In some embodiments, the genetically modified non-human animal providedherein (e.g., the rodent, e.g., the rat or the mouse) is animmunocompetent animal (e.g., immunocompetent rat or mouse).

In another aspect, disclosed herein are methods of assessing whether anantibody comprising a human Fc region can activate classical complementpathway by utilizing a genetically engineered non-human animal (e.g., arodent such as a mouse or rat) expressing a humanized C1q proteindisclosed herein. In some embodiments, the method comprises (a)providing a cell expressing an antigen of interest on the cell surface,a candidate antibody comprising a human Fc region and directed to theantigen of interest, and a serum sample from a genetically engineerednon-human animal expressing a humanized C1q protein; (b) mixing the cellwith the candidate antibody to allow the antibody to bind to the antigenof interest expressed on the cell surface; (c) adding the serum sampleto the cell-antibody mixture to permit binding of the humanized C1qproteins in the serum sample to antibodies bound to the antigen ofinterest on the cell; and (d) measuring cytotoxicity of the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

Unless specifically indicated (e.g., loxP, etc.), all human exonsequences are in empty boxes and all human intron sequences are indouble lines. All mouse or rat sequences are either in filled boxes(exons) or single lines (introns).

FIG. 1A is a schematic representation (not to scale) of the exemplarymethod of deleting of the mouse C1q locus comprising all three mouse C1qgenes (mouse genes are indicated with “m” before the gene label). Exonsof the three C1q genes are labeled below the diagram (e.g., E1, E2, andE3). Mouse BAC stands for bacterial artificial chromosome; BHR standsfor bacterial homologous recombination; EP stands for electroporation.HET=heterozygous; CM=chloramphenicol; lox=loxP site; pgk-Neo=neomycinselection cassette.

FIG. 1B shows a schematic representation (not to scale) of the creationof a humanized mouse C1q targeting vector, with chimeric human/mousegenes inserted by digestion/ligation and/or bacterial homologousrecombination (BHR) into the mouse BAC genes (mouse genes are indicatedwith “m” before the gene label). Exons of the three C1q genes arelabeled below the diagram (e.g., E1, E2, and E3). Several restrictionenzyme locations are indicated. CM=chloramphenicol; lox=loxP site;Ub-Hyg=hygromycin selection cassette; p=polyA tail; Spec=spectinomycin.

FIG. 1C shows a schematic representation (not to scale) of theelectroporation (EP) of a large targeting vector containing all threemouse/human chimeric C1q genes into mouse C1q KO HET ES cells. Exons ofthe three C1q genes are labeled below the diagram (e.g., E1, E2, andE3). Lox=loxP site; Ub-Hyg=hygromycin selection cassette;pgk-Neo=neomycin selection cassette; p=polyA sequence. Sequencejunctions between mouse, human, or cassette sequences are indicated witha line and a SEQ ID NO for that respective sequence below each junction.

FIG. 2A shows a schematic representation (not to scale) of the exemplarymethod of deleting of the rat C1q locus comprising all three rat C1qgenes (rat genes are indicated with “r” before the gene label). Exons ofthe three C1q genes are labeled below the diagram (e.g., E1, E2, andE3). Rat C1q BAC stands for rat bacterial artificial chromosome; BHRstands for bacterial homologous recombination; EP stands forelectroporation. HET=heterozygous; CM=chloramphenicol; loxP=loxP site;SDC-loxP-Hyg=self-deleting LoxP-Hygromycin selection cassette.

FIG. 2B shows a schematic representation (not to scale) of the creationof a humanized rat C1q cassette, with chimeric human/rat genes insertedby digestion/Gibson assembly and/or CAS9/Gibson assembly into the ratBAC genes (rat genes are indicated with “r” before the gene label).Exons of the three C1q genes are labeled below the diagram (e.g., E1,E2, and E3). Several restriction enzyme locations are indicated.CM=chloramphenicol; SDC-loxp-puro=self-deleting loxp−puromycin cassette.

FIG. 2C shows a schematic representation (not to scale) of theelectroporation (EP) of a large targeting vector containing all threerat/human chimeric C1q genes into rat C1q KO HET ES cells. Exons of thethree C1q genes are labeled below the diagram (e.g., E1, E2, and E3).SDC-loxp-puro=self-deleting loxp−puromycin cassette; p=polyA sequence.Sequence junctions between rat, human, or cassette sequences areindicated with a line and a SEQ ID NO for that respective sequence beloweach junction.

FIG. 3A shows C1qa amino acid alignments for rat (rC1qa), human (hC1QA),and mouse (mC1qa) polypeptides, with similarities being outlined andmismatches shown in lower cases. Signal peptide sequences are boxed andlabeled. The collagen triple helix repeat sequences are boxed andlabeled. The C1qa globular head domain sequences are boxed with dashedlines, and the junction of mouse/human or rat/human sequence in thechimeric polypeptides is depicted with a dashed line and indicated withan arrow.

FIG. 3B shows C1qb amino acid alignments for rat (rC1qb), human (hC1QB),and mouse (mC1qb) polypeptides, with similarities being outlined andmismatches shown in lower cases. Signal peptide sequences are boxed andlabeled. The collagen triple helix repeat sequences are boxed andlabeled. The C1qb globular head domain sequences are boxed with dashedlines, and the junction of mouse/human or rat/human sequence in thechimeric polypeptides is depicted with a dashed line and indicated withan arrow.

FIG. 3C shows C1qc amino acid alignments for rat (rC1qc), human (hC1QC),and mouse (mC1qc) polypeptides, with similarities being outlined andmismatches shown in lower cases. Signal peptide sequences are boxed andlabeled. The collagen triple helix repeat sequences are boxed andlabeled. The C1qc globular head domain sequences are boxed with dashedlines, and the junction of mouse/human or rat/human sequence in thechimeric polypeptides is depicted with a dashed line and indicated withan arrow.

FIG. 4 top panel shows the presence chimeric C1q proteins in the serumof humanized C1q mice as detected by anti-human C1q antibody. FIG. 4bottom panel shows a hemolysis assay measuring complement activitycomparing serum samples from wild-type littermate mouse (WT) andchimeric C1q mouse (1615 HO; HO=homozygous), and human serum.

FIG. 5 shows complement dependent cytotoxicity (CDC) activity mediatedby a human anti-CD20 antibody at 2 nM and a serum sample (normal humanserum, humanized C1q mouse serum, or wild type mouse serum) on Rajicells.

FIG. 6 shows results from a hemolysis assay measuring complementactivity comparing serum samples from wild-type littermate rats (“WT”),humanized C1q rats (“C1q Humin” or 100015HO; HO=homozygous), C1qknock-out rats (“C1q KO”), normal human serum (“NETS”), and C1q depletedhuman serum. Left panel: female rats; right panel, with the exception ofC1q KO rats: male rats.

FIG. 7 shows complement dependent cytotoxicity (CDC) activity mediatedby a human anti-CD20 antibody at 20 nM and a serum sample (normal humanserum, humanized C1q rat serum, or wild type rat serum) on Raji cells.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art which do not interfere withthe enzymatic manipulation.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art.

“Functional” as used herein, e.g., in reference to a functional protein,includes a protein that retains at least one biological activitynormally associated with the native protein. For example, in someembodiments, a replacement at an endogenous locus (e.g., replacement atendogenous non-human C1q loci) results in a locus that fails to expressa functional endogenous protein.

The term “operably linked” includes a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. As such, a nucleic acid sequenceencoding a protein may be operably linked to regulatory sequences (e.g.,promoter, enhancer, silencer sequence, etc.) so as to retain propertranscriptional regulation. For example, by “operably linked” is meant afunctional linkage between a nucleic acid expression control sequence(such as a promoter) and a second nucleic acid sequence, wherein theexpression control sequence directs transcription of the nucleic acidcorresponding to the second sequence. In addition, various portions ofthe humanized protein of the present disclosure may be operably linkedor fused to retain proper folding, processing, targeting, expression,and other functional properties of the protein in the cell. Unlessstated otherwise, various domains of the humanized protein of thepresent disclosure are operably linked to each other.

The term “humanized” as used in the phrases “humanized C1q allele,”“humanized C1qa allele,” “humanized C1qb allele,” “humanized C1qcallele,” “humanized C1q gene,” “humanized C1qa gene,” “humanized C1qbgene” or “humanized C1qc gene,” includes, but is not limited to,embodiments wherein all or a portion of an endogenous non-human C1q,C1qa, C1qb, and/or C1qc gene or allele is replaced by a correspondingportion of the human C1q, C1qa, C1qb, and/or C1qc gene or allele. Forexample, in some embodiments, the term “humanized” refers to thecomplete replacement of the coding region (e.g., the exons) of theendogenous non-human C1q, C1qa, C1qb, and/or C1qc gene or allele withthe corresponding coding region of the human C1q, C1qa, C1qb, and/orC1qc gene or allele, while the endogenous non-coding region(s) (such as,but not limited to, the promoter, the 5′ and/or 3′ untranslatedregion(s), enhancer elements, etc.) of the non-human animal may not bereplaced. In some embodiments, the humanized gene or allele are placedeither randomly in the genome or targeted to a particular locationwithin the genome. Thus, in some embodiments, the humanized gene orallele is placed in a location in the genome that is not the nativelocation for corresponding endogenous gene or allele, i.e., it is placedat a location other than the endogenous locus. In other embodiments, thehumanized gene or allele is placed at the endogenous locus; for example,the humanized gene or allele may replace the endogenous gene or alleleat the endogenous locus. In some embodiments, the non-human animal is arodent, such as a rat or mouse.

A “humanized protein” includes, but is not limited to, embodimentswherein all or a portion of the encoded endogenous non-human C1q, C1qa,C1qb, and/or C1qc protein is replaced by the corresponding portion ofthe human C1q, C1qa, C1qb, and/or C1qc protein. In some embodiments, a“humanized protein” can be encoded by a humanized C1q, C1qa, C1qb,and/or C1qc gene or allele but still is a fully human C1q, C1qa, C1qb,and/or C1qc protein (such as, but not limited to, the situation whereinall of the coding regions (e.g., the exons) of the endogenous non-humanC1q, C1qa, C1qb, and/or C1qc gene or allele are replaced by thecorresponding coding regions of the human C1q, C1qa, C1qb, and/or C1qcgene or allele but the endogenous non-coding region(s) (such as, but notlimited to, the promoter, the 5′ and/or 3′ untranslated region(s),enhancer elements, etc.) of the non-human animal is not replaced). Insome embodiments, the humanized protein is expressed from the humanizedgene or allele that is not at its native location in the genome, e.g.,it is not at the endogenous locus. In other embodiments, the humanizedprotein is expressed from the humanized gene or allele that is at theendogenous locus. In some embodiments, the humanized protein isexpressed from the humanized gene or allele that replaces the endogenousgene or allele at the endogenous locus. In some embodiments, thenon-human animal is a rodent, such as a rat or mouse.

The present disclosure is directed to like-for-like humanization. Forexample, a nucleotide sequence of an endogenous non-human C1q gene isoperably linked to a nucleotide sequence of a cognate human C1q gene toform a chimeric humanized gene. In some embodiments, a nucleotidesequence of an endogenous C1qa gene is operably linked to a nucleotidesequence of a human C1qa gene to form a humanized C1qa gene. In otherembodiments, a nucleotide sequence of an endogenous C1qb gene isoperably linked to a nucleotide sequence of a human C1qb gene to form ahumanized C1qb gene. In still other embodiments, a nucleotide sequenceof an endogenous C1qc gene is operably linked to a nucleotide sequenceof a human C1qc gene to form a humanized C1qc gene.

The term “chimeric” as used herein includes a sequence, e.g., nucleicacid or polypeptide sequence, where a portion of the sequence is derivedfrom one organism and a portion of the sequence is derived from adifferent organism. For example, a chimeric C1q polypeptide may comprisea sequence derived from a mouse or a rat, and another sequence derivedfrom a human C1q protein. In one embodiment, a chimeric C1q polypeptidecomprises a globular head domain or a fragment thereof of a human C1qpolypeptide, and a stalk domain and a stem domain of a cognate mouse orrat C1q polypeptide.

The term “locus” as in “C1q locus” refers to the location of the genomicDNA comprising a C1q coding region. For example, a C1qa locus refers tothe location of the genomic DNA comprising the C1qa coding region; aC1qb locus refers to the location of the genomic DNA comprising the C1qbcoding region; and a C1qc locus refers to the location of the genomicDNA comprising the C1qc coding region. A reference to “a C1q locus”means any one of C1qa, C1qb or C1qc locus. Other sequences may beincluded in a C1q locus that have been introduced for the purposes ofgenetic manipulation, e.g., selection cassettes, restriction sites, etc.

Other definitions and meaning of various terms used throughout thisspecification and the claims are included throughout in the relevantsections.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application. The referencesdisclosed are also individually and specifically incorporated byreference herein in their entireties for the material contained in themthat is discussed in the sentence in which the reference is relied upon.

B. Compositions

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed, whilespecific reference to each various individual and collectivecombinations and permutations of these components may not be explicitlylisted, each is specifically contemplated and described herein. Forexample, if a particular chimeric C1qa, C1qb, and/or C1qc nucleic acidor polypeptide is disclosed and discussed and a number of modificationsthat can be made to a number of molecules including the chimeric C1qa,C1qb, and/or C1qc nucleic acid or polypeptide are discussed,specifically contemplated is each and every combination and permutationof chimeric C1qa, C1qb, and/or C1qc nucleic acid or polypeptide and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

C. Polypeptides

The present disclosure provides novel chimeric mammalian C1qpolypeptides, nucleic acids encoding said polypeptides, and geneticallymodified non-human mammals that comprise said nucleic acids and canexpress the chimeric C1q polypeptides.

As used herein, “C1q” (e.g., as in “C1q protein” or “C1q complex”), isone portion of the C1 complex which, along with C1r and C1s, initiatesactivation of the complement pathway. C1q is the first component of theclassical complement pathway, required for clearance of pathogens,apoptotic bodies and possibly tumor cells (Ghai R et al., Immunobiology.2007; 212(4-5):253-66; Lu J H et al., Cell Mol Immunol. 2008 February;5(1):9-21). The complement pathway is a part of the innate immunesystem. Activation of the complement pathway can result in direct lysisof antigen, recruitment of phagocytes and macrophages to the antigenicsite, opsonization of the antigen by phagocytes, and cytokine secretion.

Human/mouse/rat C1q protein is composed of polypeptides encoded by threegenes, C1qa, C1qc, and C1qb genes, which are arranged tandemly 5′-3′ inthe order A-C-B (Petry F et al., Immunogenetics. 1996; 43(6):370-6).These polypeptides are also referred to herein as a “C1q polypeptide”,which can be a C1qa polypeptide, a C1qb polypeptide or a C1qcpolypeptide. Each of the C1qa, C1qb and C1qc polypeptide chains has anN-terminus region that includes a stalk (or neck) domain containing acysteine residue followed by a collagen-like domain (stem domain), andan C-terminus region (globular head domain). The N-terminal region isalso referred to herein as “N-terminal stalk-stem region”, and theC-terminal region is also referred to as the “globular head domain”.Human C1q protein (approx. 410 kDa) resembles a bouqet-like structureassembled from six collagenous stems, each ending with a globular head.Each stem/globular head is made up of three polypeptide chains (C1qA,C1qB and C1qC) for a total of 18 polypeptides (six A-, six B-, and sixC-chains making up one C1q molecule (Reid K B Biochem Soc Trans. 1983January; 11(1):1-12)).

C1q is expressed by splenic macrophages and dendritic cells in bothhumans and rodents (Castellano G et al., Blood. 2004 May 15;103(10):3813-20). C1q is secreted and found in human circulation atapproximately 100 ug/ml, and at similar levels in mice (Dillon S P etal., Biotechnol J. 2009 August; 4(8): 1210-1214; Yonemasu K et al., IntArch Allergy Appl Immunol. 1988; 86(1):97-101). The protein belongs to agroup of defense collagens which recognize pathogen-associated molecularpatterns (PAMPs), where the C-terminus/globular head of C1q recognizesthe CH3 domain of IgM; CH2 domain of IgG; beta-amyloid; poly-anionsincluding DNA; C-reactive protein; serum amyloid P, as well as PAMPsassociated with LPS, viruses, and prions (Dunkelberger J R, Song W C,Cell Res. 2010 January; 20(1):34-50; Ghai R et al., Immunobiology. 2007;212(4-5):253-66). C1q spontaneously assembles with C1s-C1r-C1r-C1stetramer to form C1 macrocomplex. The C1 macrocomplex is a pentamer ofthree proteins comprising one C1q complex, and two each of C1r and C1s,where C1r and C1s associate with the stalk-stem domains of C1q. C1r andC1s are regulated by serpin protease inhibitor family member C1inhibitor (C1INH) (Beinrohr L et al., Trends Mol Med. 2008 December;14(12):511-21). C1q also binds receptors including CD93, DC-SIGN andCR1/CD35 (Hosszu K K et al., Blood. 2012 Aug. 9; 120(6):1228-36; BohlsonS S at al., Mol Immunol. 2007 January; 44(1-3):33-43). By binding PAMPs,C1q opsonizes and facilitates pathogen clearance and can enhancephagocytosis of target particles sub-optimally opsonized with C3b/C4b orIgG, via CR1 or FcgR, respectively (Bobak D A et al., J Immunol. 1987Feb. 15; 138(4):1150-6). Thus, C1q activates the classical complementpathway, resulting in both formation of the membrane attack complex(target lysis) and also generation of activation fragments C3a and C5a(Bohlson S S at al., Mol Immunol. 2007 January; 44(1-3):33-43). Lastly,C1q mediates clearance of immune complexes, as well as cells undergoingapoptosis and cells blebs by recognizing apoptotic cell-associatedmolecular patterns.

To activate the classical complement pathway, C1q binds to the pathogensurface or Fc domain of antibodies through its six globular heads, whichresults in activation of C1r and C1s serine proteases, leading tocleavage of downstream complement components and ultimately complementactivation, deposition and cell lysis through the membrane attackcomplex (see Reid K B Biochem Soc Trans. 1983 January; 11(1):1-12)).

Exemplary sequences and GenBank Accession Numbers of human, mouse andrat C1qa, C1qb, and C1qc are presented in Tables 1, 2, and 8 below, andin FIG. 3A (C1qa), FIG. 3B (C1qb), and FIG. 3C (C1qc).

As it is C1q that recognizes either the Fc domain of antibodies orantigen directly, it is C1q that is key to providing a humanizedcomplement system. As the globular head domain of C1q (often abbreviatedas gC1q) is what recognizes the human antibody or the human pathogen, incertain embodiments provided herein, the globular head domain of C1q wasengineered such that it more readily recognizes these molecules. Incertain embodiments, the globular head domain of C1q is human while theremainder of the protein is non-human. In such embodiment, the non-humananimals provided herein retain the portion of the protein that is knownto interact with the C1r/C1s and the remainder of the complement system(e.g., the stem and stalk portion of the protein). Thus, in theembodiments provided herein, the non-human animal expressing C1qharboring a globular head domain that is substantially human and anN-terminal stalk-stem region that is substantially non-human is usefulin assessing the requirement for complement system as an effectormechanism of action of a therapeutic molecule, e.g., an antibody. Inembodiments provided, said non-human animal is also useful to study theeffectiveness of the therapeutic treatment if the complement system isengaged by the antibody. In embodiments provided, said non-human animalsare also useful as an in vivo model to test the efficacy of fully humantherapeutic antibodies, e.g., bispecific antibodies, designed forinfectious disease indications.

Thus, in one aspect, disclosed herein are chimeric mammalian C1qpolypeptides (such as, for example chimeric mammalian C1qa, C1qb and/orC1qc polypeptides).

In some embodiments, the chimeric C1q polypeptide provided hereincomprises a human C terminal region that is responsible for recognitionof immunoglobulin Fc domain, or responsible for recognizingpathogen-associated molecular patterns (PAMPs).

In some embodiments, the chimeric C1q polypeptide provided hereincomprises a human globular head domain or a fragment thereof.

In some embodiments, a chimeric C1q polypeptide provided hereincomprises a globular head domain that is substantially human. By “aglobular head domain that is substantially human”, it is meant that theglobular head domain in a chimeric (humanized) C1q polypeptide issubstantially identical to the globular head domain of a human C1qpolypeptide. By “substantially identical”, it refers to, (i) in someembodiments, a globular head domain that is at least 90%, 95%, 98%, 99%or 100% identical in sequence to the globular head domain of a human C1qpolypeptide; (ii) in other embodiments, a globular head domain thatdiffers from the globular head domain of a human C1q polypeptide by notmore than 5, 4, 3, 2 or 1 amino acid(s); (iii) in still otherembodiments, a globular head domain that differs from the globular headdomain of a human C1q polypeptide only at the N- or C-terminal portionof the domain, e.g., by having the same length but with one or more(e.g., 1, 2, 3, 4, or 5, but not more than 5) amino acid substitutions(such as a conservative substitution) within the N- or C-terminalportion of the globular head domain (e.g., within the 5-10 amino acidsat the N- or C-terminus of the globular head domain); and/or (iv) inother embodiments, a globular head domain that is shorter or longer thanthe globular head domain of a human C1q polypeptide by 1, 2, 3, 4, 5 butnot more than 5 amino acids at either the N- or C-terminus of thedomain. In some embodiments, a globular head domain substantiallyidentical to the globular head domain of a human C1q polypeptide differsfrom the human globular head domain by not more than 1, 2, or 3 aminoacids within the N-terminal portion (e.g., within the 5-10 amino acidsfrom the N-terminus) of the domain; and in certain such embodiments, thedifference comprises a substitution(s) of an amino acid in the humanglobular head domain with the amino acid at the corresponding positionfrom a cognate non-human (e.g., a mouse or rat) globular head domain.For example, disclosed herein in FIG. 3A is a chimeric C1qa polypeptidethat has a globular head domain that is substantially human—the globulardomain of this chimeric C1qa polypeptide differs from the globular headdomain of a human C1qa of SEQ ID NO: 4 by only one amino acid at thethird position from the N-terminus of the globular head domain (“K” inhuman, and “R” in the chimeric, mouse and rat C1qa polypeptides).

In some embodiments, the chimeric C1q polypeptide comprises an Nterminal stalk-stem region that is responsible for recognizing non-human(e.g., endogenous rodent) C1s and C1r and/or other non-human (e.g.,endogenous rodent) components of the complement pathway.

In some embodiments, the chimeric C1q polypeptide comprises a non-humanstalk-stem region. In some embodiments, the chimeric C1q polypeptidecomprises a non-human stalk domain. In some embodiments, the chimericC1q polypeptide comprises a non-human collagen triple helix domain. Insome embodiments, the chimeric C1q polypeptide comprises a non-human Nterminal region comprising non-human stem and non-human stalk domains.

In some embodiments, a chimeric C1q polypeptide provided hereincomprises an N-terminal stalk-stem region that is substantiallynon-human. By “an N-terminal stalk-stem region that is substantiallynon-human”, it is meant that the N-terminal stalk-stem region of achimeric C1q polypeptide is substantially identical to the correspondingN-terminal stalk-stem region of a non-human (e.g., a rodent) C1qpolypeptide. By “substantially identical”, it is meant (i) in someembodiments, an N-terminal stalk-stem region that is at least 90%, 95%,95%, 99% or 100% identical in sequence with the N-terminal stalk-stemregion of a non-human C1q polypeptide; (ii) in other embodiments, anN-terminal stalk-stem region that differs from the N-terminal stalk-stemregion of a non-human C1q polypeptide by not more than 5, 4, 3, 2 or 1amino acid(s); (iii) in still other embodiments, an N-terminalstalk-stem region that differs from the N-terminal stalk-stem region ofa non-human C1q polypeptide only at the C-terminus, e.g., by having thesame length but with one or more amino acid substitutions of the 5-10amino acids from the C-terminus of the stalk-stem region; and/or (iv) inother embodiments, an N-terminal stalk-stem region that is shorter orlonger than the N-terminal stalk-stem region of a non-human C1qpolypeptide by 1, 2, 3, 4, or 5 but not more than 5 amino acids at theN- or C-terminus of the region. In specific embodiments, the N-terminalstalk-stem region of a chimeric C1q polypeptide is identical to theN-terminal stalk-stem region of a non-human (e.g., rodent) C1qpolypeptide.

In one embodiment, a human C1qa polypeptide comprises the amino acidsequence as set forth in SEQ ID NO: 4. In another embodiment, a humanC1qa polypeptide comprises the amino acid sequence as set forth inGenBank Accession No. NP_001334394.1. In one embodiment, a human C1qbpolypeptide comprises the amino acid sequence as set forth in SEQ ID NO:5. In another embodiment, a human C1qb polypeptide comprises the aminoacid sequence as set forth in GenBank Accession No. NP_000482.3. In oneembodiment, a human C1qc polypeptide comprises the amino acid sequenceas set forth in SEQ ID NO: 6. In another embodiment, a human C1qcpolypeptide comprises the amino acid sequence as set forth in GenBankAccession No. NP_001334548.1.

In one embodiment, a mouse C1qa polypeptide comprises the amino acidsequence as set forth in SEQ ID NO: 1. In another embodiment, a mouseC1qa polypeptide comprises the amino acid sequence as set forth inGenBank Accession No. NP_031598.2. In one embodiment, a mouse C1qbpolypeptide comprises the amino acid sequence as set forth in SEQ ID NO:2. In another embodiment, a mouse C1qb polypeptide comprises the aminoacid sequence as set forth in GenBank Accession No. NP_033907.1. In oneembodiment, a mouse C1qc polypeptide comprises the amino acid sequenceas set forth in SEQ ID NO: 3. In another embodiment, a mouse C1qcpolypeptide comprises the amino acid sequence as set forth in GenBankAccession No. NP_031600.2.

In one embodiment, a rat C1qa polypeptide comprises the amino acidsequence as set forth in SEQ ID NO: 7. In another embodiment, a rat C1qapolypeptide comprises the amino acid sequence as set forth in GenBankAccession No. NP_001008515.1. In one embodiment, a rat C1qb polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO: 8. Inanother embodiment, a rat C1qb polypeptide comprises the amino acidsequence as set forth in GenBank Accession No. NP_062135.1. In oneembodiment, a rat C1qc polypeptide comprises the amino acid sequence asset forth in SEQ ID NO: 9. In another embodiment, a rat C1qc polypeptidecomprises the amino acid sequence as set forth in GenBank Accession No.NP_001008524.1.

Thus, in one aspect, the chimeric mammalian C1q polypeptide is achimeric C1qa polypeptide.

In some embodiments, the chimeric C1qa polypeptide comprises a humanglobular head domain or a fragment thereof. In one embodiment, thepolypeptide is a chimeric C1qa polypeptide comprising amino acids122-222 of the human C1qa polypeptide as set forth in SEQ ID NO: 4 (forexample, a chimeric mammalian C1qa polypeptide comprising amino acids122-235 or 112-245 of the human C1qa polypeptide).

In some embodiments, the chimeric C1qa polypeptide comprises a globularhead domain that is substantially human (i.e., a globular head domainsubstantially identical to the globular head domain of a human C1qapolypeptide). In one embodiment, a human C1qa polypeptide comprises theamino acid sequence of SEQ ID NO: 4, and amino acids 108-245 of SEQ IDNO: 4 constitute the globular head domain of the human C1qa polypeptideof SEQ ID NO: 4 (see FIG. 3A). Thus, in some embodiments, a chimericC1qa polypeptide comprises a globular head domain that is substantiallyidentical to the human C1qa globular head domain represented by aminoacids 108-245 of SEQ ID NO: 4. In a specific embodiment, the globularhead domain of a chimeric C1qa polypeptide is represented by amino acids108-245 of SEQ ID NO: 10, such domain differing from the human C1qaglobular head domain represented by amino acids 108-245 of SEQ ID NO: 4in one amino acid (the third amino acid residue being “R” as in mouseand rat C1qa, instead of “K” in human C1qa). In another specificembodiment, a chimeric C1qa polypeptide comprises a globular head domainthat is identical to the human C1qa globular head domain represented byamino acids 108-245 of SEQ ID NO: 4.

In some embodiments, a chimeric C1qa polypeptide comprises non-humanC1qa stalk and/or stem domains. Thus, in some embodiments, a chimericmammalian C1qa polypeptide comprises a non-human sequence which is amouse sequence comprising at least amino acids 33-102, 30-102, 25-105,23-107 or 23-111 of the mouse C1qa polypeptide set forth in SEQ IDNO: 1. In other embodiments, the chimeric mammalian C1qa polypeptidecomprises a non-human sequence which is a rat sequence comprising atleast amino acids 33-102, 30-102, 25-105, 23-107 or 23-111 of the ratC1qa polypeptide set forth in SEQ ID NO: 7.

In some embodiments, the chimeric C1qa polypeptide comprises anN-terminal stalk-stem region that is substantially non-human, i.e., anN-terminal stalk-stem region that is substantially identical to theN-terminal stalk-stem region of a non-human C1qa polypeptide. In certainembodiments, the chimeric C1qa polypeptide comprises an N-terminalstalk-stem region that is substantially identical to the N-terminalstalk-stem region of a mouse C1qa polypeptide. In one embodiment, themouse C1qa polypeptide comprises the amino acid sequence of SEQ ID NO: 1(FIG. 3A), and amino acids 23-107 of SEQ ID NO: 1 constitute theN-terminal stalk-stem region of the mouse C1qa polypeptide of SEQ ID NO:1 (FIG. 3A). Thus, in some embodiments, a chimeric C1qa polypeptidecomprises an N-terminal stalk-stem region that is substantiallyidentical to the mouse C1qa N-terminal stalk-stem region represented byamino acids 23-107 of SEQ ID NO: 1. In a specific embodiment, theN-terminal stalk-stem region of a chimeric C1qa polypeptide isrepresented by amino acids 23-107 of SEQ ID NO: 1. In some embodiments,the chimeric C1qa polypeptide comprises an N-terminal stalk-stem regionthat is substantially identical to the N-terminal stalk-stem region of arat C1qa polypeptide. In one embodiment, the rat C1qa polypeptidecomprises the amino acid sequence of SEQ ID NO: 7 (FIG. 3A), and aminoacids 23-107 of SEQ ID NO: 7 constitute the N-terminal stalk-stem regionof the rat C1qa polypeptide of SEQ ID NO: 7 (FIG. 3A). Thus, in someembodiments, a chimeric C1qa polypeptide comprises an N-terminalstalk-stem region that is substantially identical to the rat C1qaN-terminal stalk-stem region represented by amino acids 23-107 of SEQ IDNO: 7. In a specific embodiment, the N-terminal stalk-stem region of achimeric C1qa polypeptide is represented by amino acids 23-107 of SEQ IDNO: 7.

In some embodiments, the chimeric C1qa polypeptide comprises a globularhead domain that is substantially human and an N-terminal stalk-stemregion that is substantially non-human. For example, disclosed herein isa chimeric mammalian C1qa polypeptide, wherein the chimeric mammalianC1qa polypeptide comprises at least amino acids 23-245 of thepolypeptide set forth in SEQ ID NO: 10 (mouse/human) or at least aminoacids 23-245 of the polypeptide set forth in SEQ ID NO:55 (rat/human).In one aspect, the chimeric C1qa polypeptide is or comprises SEQ ID NO:10 or SEQ ID NO: 55.

In another aspect, the chimeric mammalian C1q polypeptide is a C1qbpolypeptide.

In some embodiments, the chimeric C1qb polypeptide comprises a humanC1qb globular head or a fragment thereof. In one embodiment, thepolypeptide is a chimeric C1qb polypeptide comprising amino acids125-233 of the human C1qb polypeptide as set forth in SEQ ID NO: 5 (suchas, for example, amino acids 120-250 or 118-251 of the human C1qbpolypeptide).

In some embodiments, the chimeric C1qb polypeptide comprises a globularhead domain that is substantially human (i.e., a globular head domainsubstantially identical to the globular head domain of a human C1qbpolypeptide). In one embodiment, a human C1qb polypeptide comprises theamino acid sequence of SEQ ID NO: 5 (FIG. 3B), and amino acids 115-251of SEQ ID NO: 5 constitute the globular head domain of the human C1qbpolypeptide of SEQ ID NO: 5 (FIG. 3B). Thus, in some embodiments, achimeric C1qb polypeptide comprises a globular head domain that issubstantially identical to the human globular head domain represented byamino acids 115-251 of SEQ ID NO: 5. In a specific embodiment, theglobular head domain of a chimeric C1qb polypeptide is represented byamino acids 115-251 of SEQ ID NO: 11, such domain differing from thehuman C1qb globular head domain represented by amino acids 115-251 ofSEQ ID NO: 5 in one amino acid (the first amino acid residue being “G”as in mouse C1qb, instead of “K” in human C1qb). In another specificembodiment, a chimeric C1qb polypeptide comprises a globular head domainthat is identical to the human C1qb globular head domain represented byamino acids 115-251 of SEQ ID NO: 5.

In some embodiments, a chimeric C1qb polypeptide comprises non-humanC1qb stalk and/or stem domains. In some embodiments, the chimericmammalian C1qb polypeptide comprises a non-human mammal sequence whichis a mouse sequence comprising at least amino acids 32-105, 27-105,27-110, 26-114, or 26-117 of the mouse C1qb polypeptide set forth in SEQID NO: 2. In other embodiments, the chimeric mammalian C1qb polypeptidecomprises a non-human mammal sequence which is a rat sequence comprisingat least amino acids 32-105, 27-105, 27-110, 26-114, or 26-117 of therat C1qb polypeptide set forth in SEQ ID NO: 8.

In some embodiments, the chimeric C1qb polypeptide comprises anN-terminal stalk-stem region that is substantially non-human, i.e., anN-terminal stalk-stem region that is substantially identical to theN-terminal stalk-stem region of a non-human C1qb polypeptide. In certainembodiments, the chimeric C1qb polypeptide comprises an N-terminalstalk-stem region that is substantially identical to the N-terminalstalk-stem region of a mouse C1qb polypeptide. In one embodiment, themouse C1qb polypeptide comprises the amino acid sequence of SEQ ID NO: 2(FIG. 3B), and amino acids 26-114 of SEQ ID NO: 2 constitute theN-terminal stalk-stem region of the mouse C1qb polypeptide of SEQ ID NO:2 (FIG. 3B). Thus, in some embodiments, a chimeric C1qb polypeptidecomprises an N-terminal stalk-stem region that is substantiallyidentical to the mouse C1qb N-terminal stalk-stem region represented byamino acids 26-114 of SEQ ID NO: 2. In a specific embodiment, theN-terminal stalk-stem region of a chimeric C1qb polypeptide isrepresented by amino acids 26-114 of SEQ ID NO: 2. In some embodiments,the chimeric C1qb polypeptide comprises an N-terminal stalk-stem regionthat is substantially identical to the N-terminal stalk-stem region of arat C1qb polypeptide. In one embodiment, the rat C1qb polypeptidecomprises the amino acid sequence of SEQ ID NO: 8 (FIG. 3B), and aminoacids 26-114 of SEQ ID NO: 8 constitute the N-terminal stalk-stem regionof the rat C1qb polypeptide of SEQ ID NO: 8 (FIG. 3B). Thus, in someembodiments, a chimeric C1qb polypeptide comprises an N-terminalstalk-stem region that is substantially identical to the rat C1qbN-terminal stalk-stem region represented by amino acids 26-114 of SEQ IDNO: 8. In a specific embodiment, the N-terminal stalk-stem region of achimeric C1qb polypeptide is represented by amino acids 26-114 of SEQ IDNO: 8.

In some embodiments, the chimeric C1qb polypeptide comprises a globularhead domain that is substantially human and an N-terminal stalk-stemregion that is substantially non-human. For example, disclosed herein isa chimeric mammalian C1qb polypeptide, wherein the polypeptide comprisesat least amino acids 26-251 of the polypeptide set forth in SEQ ID NO:11 (mouse/human) or at least amino acids 26-251 of the polypeptide setforth in SEQ ID NO: 56 (rat/human). In one aspect, the chimeric C1qbpolypeptide is or comprises SEQ ID NO: 11 or SEQ ID NO: 56.

In another aspect, the chimeric mammalian C1q polypeptide is a chimericC1qc polypeptide.

In some embodiments, the chimeric C1qc polypeptide comprises a humanC1qc globular head or a fragment thereof. In one embodiment, thepolypeptide is a chimeric C1qc polypeptide comprising amino acids118-234 of the human C1qc polypeptide as set forth in SEQ ID NO: 6 (forexample, a chimeric mammalian C1qc polypeptide comprising amino acids114-245 of the human C1qc polypeptide set forth in SEQ ID NO:6).

In some embodiments, the chimeric C1qc polypeptide comprises a globularhead domain that is substantially human (i.e., a globular head domainsubstantially identical to the globular head domain of a human C1qcpolypeptide). In one embodiment, the human C1qc polypeptide comprisesthe amino acid sequence of SEQ ID NO: 6 (FIG. 3C), and amino acids113-245 of SEQ ID NO: 6 constitute the globular head domain of the humanC1qc polypeptide of SEQ ID NO: 6 (FIG. 3C). Thus, in some embodiments, achimeric C1qc polypeptide comprises a globular head domain that issubstantially identical to the human C1qc globular head domainrepresented by amino acids 113-245 of SEQ ID NO: 6. In a specificembodiment, the globular head domain of a chimeric C1qc polypeptide isidentical to the human C1qc globular head domain represented by aminoacids 113-245 of SEQ ID NO: 6.

In some embodiments, a chimeric C1qc polypeptide comprises non-humanC1qc stalk and/or stem domains. For example, disclosed herein is achimeric mammalian C1qc polypeptide wherein the non-human mammalsequence is a mouse sequence comprising at least amino acids 31-111,30-113, or 30-114 of the mouse C1qc polypeptide set forth in SEQ ID NO:3; or the chimeric mammalian C1qc polypeptide wherein the non-humanmammal sequence is a rat sequence comprising at least amino acids33-113, 32-115, or 32-116 of the rat C1qc polypeptide set forth in SEQID NO: 9.

In some embodiments, the chimeric C1qc polypeptide comprises anN-terminal stalk-stem region that is substantially non-human, i.e., anN-terminal stalk-stem region that is substantially identical to theN-terminal stalk-stem region of a non-human C1qc polypeptide. In certainembodiments, the chimeric C1qc polypeptide comprises an N-terminalstalk-stem region that is substantially identical to the N-terminalstalk-stem region of a mouse C1qc polypeptide. In one embodiment, themouse C1qc polypeptide comprises the amino acid sequence of SEQ ID NO: 3(FIG. 3C), and amino acids 30-113 of SEQ ID NO: 3 constitute theN-terminal stalk-stem region of the mouse C1qc polypeptide of SEQ ID NO:3 (FIG. 3C). Thus, in some embodiments, a chimeric C1qc polypeptidecomprises an N-terminal stalk-stem region that is substantiallyidentical to the mouse C1qc N-terminal stalk-stem region represented byamino acids 30-113 of SEQ ID NO: 3. In a specific embodiment, theN-terminal stalk-stem region of a chimeric C1qc polypeptide isrepresented by amino acids 30-113 of SEQ ID NO: 3. In some embodiments,the chimeric C1qc polypeptide comprises an N-terminal stalk-stem regionthat is substantially identical to the N-terminal stalk-stem region of arat C1qc polypeptide. In one embodiment, the rat C1qc polypeptidecomprises the amino acid sequence of SEQ ID NO: 9 (FIG. 3C), and aminoacids 32-115 of SEQ ID NO: 9 constitute the N-terminal stalk-stem regionof the rat C1qc polypeptide of SEQ ID NO: 9 (FIG. 3C). Thus, in someembodiments, a chimeric C1qc polypeptide comprises an N-terminalstalk-stem region that is substantially identical to the rat C1qcN-terminal stalk-stem region represented by amino acids 32-115 of SEQ IDNO: 9. In a specific embodiment, the N-terminal stalk-stem region of achimeric C1qc polypeptide is represented by amino acids 32-115 of SEQ IDNO: 9.

In some embodiments, the chimeric C1qc polypeptide comprises a globularhead domain that is substantially human and an N-terminal stalk-stemregion that is substantially non-human. For example, also disclosedherein is a chimeric mammalian C1qc polypeptide, wherein the polypeptidecomprises at least amino acids 30-246 of the polypeptide set forth inSEQ ID NO: 12 (mouse/human) or at least amino acids 32-248 of thepolypeptide set forth in SEQ ID NO: 57 (rat/human). In one aspect, thechimeric C1qc polypeptide is or comprises SEQ ID NO: 12 or SEQ ID NO:57.

In one particular aspect, disclosed herein is a chimeric C1q proteincomprising one or more of the chimeric C1q polypeptides describedherein. For example, disclosed herein is a chimeric C1q protein, whereinthe protein comprises at least one, two, three, four, five, or sixchimeric C1qa, one, two, three, four, five, or six chimeric C1qb, and/orone, two, three, four, five, or six chimeric C1qc polypeptide. Thus, insome embodiments, the chimeric protein comprises six of each chimericC1qa polypeptide, chimeric C1qb polypeptide, and chimeric C1qcpolypeptide.

The disclosed C1q polypeptides are chimeric polypeptides comprising parthuman and part non-human amino acid structure. It is understood andherein contemplated that the human and non-human portions of thedisclosed chimeric polypeptides are linked, fused, or otherwisechemically joined in such a manner as to retain functionality of the C1qpolypeptide. As used herein, “function” and “functionality” refer to theability to carry out the duties of the native molecule. For C1qa, C1qband C1qc, functionality includes the ability to form dimers (for C1qaand C1qb heterodimers and for C1qc homodimers), the ability for eachportion of the chimeric polypeptide to assume proper folding, theability to assemble as a trimer of dimers (two C1qa and C1qbheterodimers and 1 C1qc homodimer per trimer) forming a C1q complex, theability to form a pentamer C1 complex with C1r and C1s, the ability torecognize an Fc domain of an antibody or PAMPs, and the ability initiatethe classical complement pathway. The assembly of a C1q protein has beendescribed (see, e.g., Lu et al. (Cellular & Mol. Immunol. 2008, 5(1):9-21), especially FIG. 1). C1qa and C1qb polypeptide chains dimerizethrough a disulphide bond at the N-terminal end and two C1qc chains formhomodimers through similar disulphide bonding. A C1qa-C1qb dimer and asingle C1qc chain form a triple helix and the other C1qc-chain in aC1qc-C1qc dimer trimerizes with another C1qa-C1qb dimer forming twotriple helices linked by the disulphide bond between the two C1qcchains. Three such structures form a C1q protein molecule throughN-terminal association.

In a particular aspect, disclosed herein is a chimeric mammalian C1qa,C1qb, and/or C1qc polypeptides, wherein the polypeptide disclosed hereinfurther comprises a human, a mouse, or a rat C1qa, C1qb, and/or C1qcsignal sequence, respectively. Exemplary human, mouse, and rat C1qa,C1qb, and C1qc signal sequence are shown in FIGS. 3A, 3B, and 3C,respectively.

It is understood and herein contemplated that any of the disclosedchimeric polypeptides can be expressed in a non-human animal. Thus,encompassed by the disclosure is a genetically modified non-humananimal, e.g., rodent, e.g., mouse or rat, expressing a chimeric C1qprotein(s) described herein or chimeric C1q protein(s) comprisingvariants, e.g., conservative amino acid substitutions, of the amino acidsequence(s) described herein.

Thus, the chimeric polypeptide can be one of the numerous variants ofthe chimeric C1qa, C1qb, and/or C1qc polypeptide that are known andherein contemplated. In addition to the known functional C1qa, C1qb,and/or C1qc species and strain variants, there are derivatives of theC1qa, C1qb, and/or C1qc polypeptides which also function in thedisclosed methods and compositions. Protein variants and derivatives arewell understood to those of skill in the art and can involve amino acidsequence modifications. For example, amino acid sequence modificationstypically fall into one or more of three classes: substitutional,insertional or deletional variants. These types of modifications andmolecular techniques to achieve them are known in the art.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final construct. These modifications must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. Theterm “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). Conservative amino acidsubstitutions may be achieved by modifying a nucleotide sequence so asto introduce a nucleotide change that will encode the conservativesubstitution. In general, a conservative amino acid substitution willnot substantially change the functional properties of interest of aprotein, for example, the ability of C1q complex to bind immunoglobulinor activate the complement pathway. Examples of groups of amino acidsthat have side chains with similar chemical properties include aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be a substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. ((1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45), hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

Substantial changes in function are made by selecting substitutions thatare less conservative than those listed above, i.e., selecting residuesthat differ more significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,for example as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the protein properties will be those in which(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, (e) by increasing thenumber of sites for sulfation and/or glycosylation.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NO: 1 sets forth a particular sequence of mouse C1qapolypeptide, SEQ ID NO: 2 sets forth a particular sequence of mouse C1qbpolypeptide, SEQ ID NO: 3 sets forth a particular sequence of mouse C1qcpolypeptide, SEQ ID NO: 4 sets forth a particular sequence of human C1qapolypeptide, SEQ ID NO: 5 sets forth a particular sequence of human C1qbpolypeptide, SEQ ID NO: 6 sets forth a particular sequence of human C1qcpolypeptide, SEQ ID NO: 7 sets forth a particular sequence of rat C1qapolypeptide, SEQ ID NO: 8 sets forth a particular sequence of rat C1qbpolypeptide and SEQ ID NO: 9 sets forth a particular sequence of a ratC1qc polypeptide. Specifically disclosed are variants of these and otherproteins herein disclosed which have at least 90%, 95%, 98%, or 99%homology to the stated sequence. In some embodiments, the homologoussequences are those represented by GenBank Accession Numbers listed inTables 1, 2, and 8. Those of skill in the art readily understand how todetermine the homology of two proteins. For example, the homology can becalculated after aligning the two sequences so that the homology is atits highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99% homology to a particular sequence wherein the variants areconservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e., all nucleicacids having a sequence that encodes one particular protein sequence aswell as all nucleic acids, including degenerate nucleic acids, encodingthe disclosed variants and derivatives of the protein sequences. Thus,while each particular nucleic acid sequence may not be written outherein, it is understood that each and every sequence is in factdisclosed and described herein through the disclosed protein sequence.

D. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for examplethe chimeric C1qa, C1qb, C1qc, or any of the nucleic acids disclosedherein for making C1qa, C1qb, and/or C1qc genetically modified non-humananimals, as well as various functional nucleic acids.

Disclosed herein are isolated nucleic acids encoding a chimericmammalian C1qa, C1qb, and/or C1qc polypeptides comprising non-human andhuman nucleic acid sequences. In some embodiments, the isolated nucleicacid sequences described herein are large targeting vectors includingentire regions of the genome, and include exons, introns, and/orintergenic sequences. These may include 5′ and 3′ untranslated regions,enhancers, promoters, and other regulatory regions. In some embodiments,these regulatory elements are non-human regulatory elements, e.g.,regulatory elements of an endogenous non-human C1q gene. In otherembodiments, these regulatory elements are human regulatory elements,e.g., regulatory elements of a human C1q gene. In other embodiments, theisolated nucleic acid sequences described herein are cDNA sequences. Insome embodiments, the isolated nucleic acids, e.g., a large targetingvector, described herein may include more than a single C1q gene, e.g.,two or three genes (e.g., it may encode all three chimeric C1qa, C1qb,and C1qc genes described herein).

In one aspect, disclosed herein is an isolated nucleic acid comprisingnon-human and human nucleic acid sequences, wherein the nucleic acidencodes a chimeric mammalian C1q polypeptide (e.g., C1qa, C1qb, or C1qcpolypeptide) described hereinabove, e.g., a chimeric C1q polypeptidecomprising an N-terminal stalk-stem region that is substantiallynon-human and a globular head domain that is substantially human.

In some embodiments, an isolated nucleic acid encoding a chimericmammalian C1qa polypeptide comprises non-human and human nucleic acidsequences, wherein the human nucleic acid sequence encodes substantiallythe globular head domain of a human C1q polypeptide, and the non-humannucleic acid sequence encodes substantially the N-terminal stalk-stemregion of a cognate non-human C1q polypeptide.

By “a human C1q nucleic acid sequence encoding substantially theglobular head domain of a human C1q polypeptide”, it means a fragment ofa human C1q gene that encodes the globular head domain or a polypeptidefragment that is slightly longer or shorter than the globular domain ofthe human C1q polypeptide. By “slightly longer or shorter” is meant adifference in length of not more than 5, 4, 3, 2, or 1 amino acid.Similarly, by “a non-human C1q nucleic acid sequence encodingsubstantially the N-terminal stalk-stem region of a non-human C1qpolypeptide”, it means a fragment of a non-human C1q gene that encodesthe N-terminal stalk-stem region or a polypeptide fragment that isslightly longer or shorter than the N-terminal stalk-stem region of thenon-human C1q polypeptide.

In circumstances where a non-human C1q polypeptide and a cognate humanC1q polypeptide share common amino acids near the junction between thestalk-stem region and the globular head domain, it may not be necessaryto utilize a human C1q nucleic acid sequence that encodes precisely theglobular head domain of the human C1q polypeptide. It is possible to usea nucleic acid sequence of a human C1q gene that encodes substantiallythe globular head domain of the human C1q polypeptide, in operablelinkage to a nucleic acid that encodes substantially the stalk-stemregion of the non-human animal C1q polypeptide, such that the chimericC1q polypeptide includes a globular head domain that is substantially orfully identical to the globular head domain of the human C1qpolypeptide, and a stalk-stem region that is substantially or fullyidentical to stalk-stem region of the non-human C1q polypeptide.Similarly, in circumstances where a non-human C1q polypeptide and ahuman C1q polypeptide share common amino acids near the C-terminus ofthe globular head domain, it may not be necessary to utilize a human C1qnucleic acid that encodes precisely the globular head domain of thehuman C1q polypeptide. It is possible to insert a slightly shorternucleic acid of a human C1q gene that encodes a polypeptide slightlyshorter than the globular head domain of the human C1q polypeptide, inoperable linkage to a non-human nucleic acid that encodes the remainderamino acids at the C-terminus of the globular head domain, such that thechimeric C1q polypeptide includes a globular head domain that is stillsubstantially or fully identical to the globular head domain of thehuman C1q polypeptide.

In one aspect, disclosed herein is an isolated nucleic acid encoding achimeric mammalian C1qa polypeptide, wherein the human nucleic acidsequence encodes substantially the globular head domain of a human C1qapolypeptide, e.g., encodes the globular head domain of a human C1qapolypeptide or a fragment thereof. For example, in one aspect, disclosedherein is an isolated nucleic acid encoding a chimeric mammalian C1qapolypeptide, wherein the nucleic acid comprises at least a nucleic acidsequence encoding amino acids 122-222 of the human C1qa polypeptide asset forth in SEQ ID NO: 4. For example, the nucleic acid can comprise anucleic acid sequence (e.g., a portion of human C1qa exon 3) encodingamino acids 122-235 or 112-245 of the human C1qa polypeptide as setforth in SEQ ID NO: 4.

The disclosed nucleic acid encoding chimeric mammalian C1qa polypeptidemay further comprise non-human nucleic acid sequences (such as, forexample, a nucleic acid sequence encoding substantially the N-terminalstalk-stem region of a non-human C1qa polypeptide). Thus, also disclosedherein are isolated nucleic acids, wherein the nucleic acid sequencecomprises at least a nucleotide sequence encoding amino acids 33-102,30-102, 25-105, 23-107 or 23-111 of the mouse C1qa polypeptide set forthin SEQ ID NO: 1, or at least a nucleotide sequence encoding amino acids33-102, 30-102, 25-105, 23-107 or 23-111 of the rat C1qa polypeptide setforth in SEQ ID NO: 7. In a particular aspect, disclosed herein is anisolated nucleic acid, wherein the isolated nucleic acid encodes atleast amino acids 23-245 of the polypeptide set forth in SEQ ID NO: 10(mouse/human) or at least amino acids 23-245 of the polypeptide setforth in SEQ ID NO: 55 (rat/human). In one aspect, the isolated nucleicacid encodes a functional chimeric mammalian C1qa polypeptide which isor comprises SEQ ID NO: 10 or SEQ ID NO: 55.

Also disclosed herein is an isolated nucleic acid encoding a chimericmammalian C1qb polypeptide comprising a nucleic acid sequence encodingsubstantially the globular head domain of a human C1qb polypeptide,e.g., encoding the globular head domain of a human C1qb polypeptide or afragment thereof. For example, the nucleic acid encoding the chimericmammalian C1qb polypeptide comprises at least a nucleic acid sequenceencoding amino acids 125-233 of the human C1qb polypeptide as set forthin SEQ ID NO: 5. For example, the nucleic acid can comprise a nucleicacid sequence (e.g., a portion of human C1qb exon 3) encoding aminoacids 118-251 of human C1qb polypeptide as set forth in SEQ ID NO: 5.

The disclosed nucleic acid encoding chimeric mammalian C1qb polypeptidemay further comprise non-human nucleic acid sequences (such as, forexample, a nucleic acid sequence encoding substantially the N-terminalstalk-stem region of a non-human C1qb polypeptide). Thus, also disclosedherein is an isolated nucleic acid, wherein the nucleic acid sequencecomprises at least a nucleotide sequence encoding amino acids 32-105,27-105, 27-110, 26-114, or 26-117 of the mouse C1qb polypeptide setforth in SEQ ID NO: 2, or wherein the nucleic acid sequence comprises atleast a nucleotide sequence encoding amino acids 32-105, 27-105, 27-110,26-114, or 26-117 of the rat C1qb polypeptide set forth in SEQ ID NO: 8.In a particular aspect, disclosed herein is an isolated nucleic acid,wherein the isolated nucleic acid encodes at least amino acids 26-251 ofthe polypeptide set forth in SEQ ID NO: 11 (mouse/human) or wherein theisolated nucleic acid encodes at least amino acids 26-251 of thepolypeptide set forth in SEQ ID NO: 56 (rat/human). In one aspect, theisolated nucleic acid encodes a functional chimeric mammalian C1qbpolypeptide which is or comprises SEQ ID NO: 11 or SEQ ID NO: 56.

Also disclosed herein is an isolated nucleic acid encoding a chimericmammalian C1qc polypeptide comprising a nucleic acid sequence encodingsubstantially the globular head domain of a human C1qc polypeptide,e.g., encoding the globular head domain of a human C1qc polypeptide or afragment thereof. In one aspect, the isolated nucleic acid encodingchimeric mammalian C1qc polypeptide, wherein the nucleic acid encodingthe chimeric mammalian C1qc polypeptide comprises at least a nucleicacid sequence encoding amino acids 118-234 of human C1qc polypeptide setforth in SEQ ID NO: 6. For example, the nucleic acid can comprise anucleic acid sequence (e.g., a portion of human C1qa exon 3) encodingamino acids 114-245 of human C1qc polypeptide as set forth in SEQ ID NO:6.

The nucleic acids encoding chimeric mammalian C1qc polypeptide mayfurther comprise non-human nucleic acid sequences (such as, for example,a nucleic acid sequence encoding the substantially the N-terminalstalk-stem region of a non-human C1qc polypeptide). Thus, also disclosedherein is an isolated nucleic acid, wherein the nucleic acid sequencecomprises at least a nucleotide sequence encoding amino acids 31-111,30-113, or 30-114 of the mouse C1qc polypeptide set forth in SEQ ID NO:3, or wherein the nucleic acid sequence comprises at least a nucleotidesequence encoding amino acids 33-113, 32-115, or 32-116 of the rat C1qcpolypeptide set forth in SEQ ID NO: 9. In a particular aspect, disclosedherein is an isolated nucleic acid, wherein the isolated nucleic acidencodes at least amino acids 30-246 of the polypeptide set forth in SEQID NO: 12 (mouse/human) or wherein the isolated nucleic acid encodes atleast amino acids 32-248 of the polypeptide set forth in SEQ ID NO: 57(rat/human). In one aspect, the isolated nucleic acid encodes afunctional chimeric mammalian C1qc polypeptide which is or comprises SEQID NO: 12 or SEQ ID NO: 57.

In some embodiments, the non-human nucleic acid sequence in an isolatednucleic acid encoding a chimeric C1q polypeptide also encodes anon-human signal peptide, e.g., the signal peptide of an endogenousnon-human C1q polypeptide.

In some embodiments, the non-human nucleic acid sequence in an isolatednucleic acid encoding a chimeric C1q polypeptide also comprises anon-human 5′ UTR region, e.g., the 5′ UTR region of an endogenousnon-human C1q gene.

In some embodiments, the human nucleic acid sequence in an isolatednucleic acid encoding a chimeric C1q polypeptide also comprises a human3′ UTR region, e.g., the 3′ UTR region of a human C1q gene.

In some embodiments, the non-human nucleic acid sequence is a genomicfragment of a non-human C1q gene which comprises a coding portion ofexon 2 (e.g., the portion that encodes amino acids of the mature form ofthe non-human C1q polypeptide) and a portion of exon 3 (e.g., theportion that encodes amino acids of the N-terminal stalk-stem region).In some embodiments, the non-human nucleic acid sequence is a genomicfragment of a non-human C1q gene comprising the entire coding portion ofexon 2 which encodes both the signal peptide and amino acids of themature form of the non-human C1q polypeptide, and the portion of exon 3that encodes amino acids of the N-terminal stalk-stem region. In someembodiments, the non-human nucleic acid sequence is a genomic fragmentof a non-human C1q gene comprising exon 1, exon 2, and the portion ofexon 3 that encodes amino acids of the N-terminal stalk-stem region,thereby encompassing the 5′ UTR of the non-human C1q gene.

In some embodiments, the human nucleic acid sequence is a genomicfragment of a human C1q gene which comprises a portion of exon 3 thatencodes the globular head domain or a fragment thereof of a human C1qpolypeptide. The human nucleic acid sequence is operably linked to thenon-human nucleic acid sequence such that the encoded chimeric C1qpolypeptide comprises an N-terminal stalk-stem region that issubstantially non-human and a globular head domain that is substantiallyhuman and is a functional C1q polypeptide.

In some embodiments, the human nucleic acid sequence is a genomicfragment of a human C1q gene comprising a 3′ portion of exon 3 thatencodes the globular head domain or a fragment thereof and includes theentire 3′ UTR of the human C1q gene.

In one particular aspect, disclosed herein is an isolated nucleic acidencoding a chimeric non-human C1q protein, wherein the nucleic acidcomprises a sequence encoding a chimeric C1qa, a chimeric C1qb, and/or achimeric C1qc. In some embodiments, one or more of the sequence(s)encoding the chimeric C1qa, C1qb, and/or C1qc comprise a sequenceencoding a human globular head domain or a fragment thereof. In someembodiments, one or more of the sequence(s) encoding the chimeric C1qa,C1qb, and/or C1qc comprise a sequence encoding non-human (e.g., rodent,e.g., rat or mouse) stem and/or stalk. In some embodiments, one or moreof the sequence(s) encoding the chimeric C1qa, C1qb, and/or C1qccomprise a sequence encoding non-human (e.g., rodent, e.g., rat ormouse) collagen triple helix domain.

Thus, in some embodiments, disclosed herein is an isolated nucleic acid,wherein the isolated nucleic acid encodes a chimeric non-human mammalC1q protein, comprising one or more of a first, second or thirdnucleotide sequences, wherein the first nucleotide sequence encodes achimeric non-human mammalian C1qa polypeptide, the second nucleotidesequence encodes a chimeric non-human mammalian C1qb polypeptide, andthe third nucleotide sequence encodes a chimeric non-human mammalianC1qc polypeptide.

In one embodiment, the disclosed isolated nucleic acids can furthercomprise a nucleotide sequence that encodes a human, a mouse or a ratC1qa, C1qb, and/or C1qc signal peptide as set forth in FIGS. 3A, 3B,and/or 3C.

Also disclosed herein is an isolated nucleic acid encoding a chimericmammalian C1q polypeptide, wherein the non-human mammal nucleic acidsequence comprises exons 1 and 2 of the non-human mammal C1qa, C1qb,and/or C1qc gene.

It is understood and herein contemplated that the disclosed nucleicacids can be incorporated into a cell to be translated and expressed.Thus, any of the disclosed nucleic acids can be cloned into the genomeof a cell for expression of the chimeric C1q polypeptide. Therefore,provided herein is a genetically modified cell comprising one or moreisolated nucleic acids of any preceding aspect.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell). In someembodiments, the cell is an ES cell. In other embodiments, the cell is adendritic cell, a fibroblast, a epithelial cell, or a primary cell. Insome embodiments, the cell is used to produce genetically modifiednon-human animals. It is further understood that some said cells can beincorporated into and used to develop a genetically modified non-humananimal comprising any of the disclosed nucleic acids which at leastencode one or more chimeric C1qa, C1qb, and/or C1qc polypeptides, andcan express said polypeptides. In some embodiments, a cell is obtainedfrom the genetically modified non-human animal provided herein. In somesuch embodiments, the cell may be a primary cell. In some embodiments,the cell may be a macrophage or a dendritic cell.

One skilled in the art would understand that in addition to the nucleicacid residues encoding humanized C1q proteins described herein, due tothe degeneracy of the genetic code, other nucleic acids may encode thepolypeptides of the present disclosure. Therefore, in addition to agenetically modified non-human animal that comprises in its genomenucleotide sequences encoding humanized C1q proteins described herein, anon-human animal that comprises in its genome nucleotide sequences thatdiffer from those described herein due to the degeneracy of the geneticcode are also provided.

There are a variety of sequences related to the protein C1q, for examplethe polypeptides C1qa, C1qb, and/or C1qc as well as chimeric C1qa, C1qb,and/or C1qc, or any of the nucleic acids disclosed herein for makingchimeric C1qa, C1qb, and/or C1qc polypeptides, all of which are encodedby nucleic acids or are nucleic acids. The sequences for the humananalogs of these genes, as well as other analogs, and alleles of thesegenes, and splice variants and other types of variants, are available ina variety of protein and gene databases, including Genbank. Those ofskill in the art understand how to resolve sequence discrepancies anddifferences and to adjust the compositions and methods relating to aparticular sequence to other related sequences.

E. Genetically Modified Humanized C1q Animals

In a further aspect, provided herein is a genetically modified non-humananimal (e.g., rodent such as mouse or rat) that expresses a chimeric,humanized C1q polypeptide (e.g., a chimeric C1qa, C1qb or C1qc) asdescribed hereinabove. Such non-human animal expresses a humanized C1qcomplex comprising the humanized C1q polypeptide.

In one aspect, disclosed herein are non-human animals (e.g., rodent suchas mouse or rat) that comprise in their genome a nucleic acid moleculeencoding a chimeric C1q polypeptide described hereinabove, e.g., achimeric C1q polypeptide (C1qa, C1qb, or C1qc) comprising an N-terminalstalk-stem region that is substantially non-human (endogenous) and aglobular head domain that is substantially human.

In some embodiments, the animal disclosed herein comprises a nucleicacid molecule encoding a chimeric C1q polypeptide (e.g., C1qa, C1qb orC1qc polypeptide) as described hereinabove, wherein the nucleic acidmolecule comprises non-human (e.g., endogenous) and human nucleic acidsequences.

In some embodiments, the nucleic acid molecule encoding a chimeric C1qpolypeptide includes a non-human (e.g., endogenous) C1q nucleic acidsequence and a cognate human C1q nucleic acid sequence, operably linkedto each other such that the nucleic acid molecule encodes a functionalC1q polypeptide. The term “cognate” is used herein in the context of achimeric molecule to indicate that the sequences in the chimericmolecule that are of different origins correspond to the same gene. Forexample, a mouse or rat C1qa sequence is linked to a human C1qa sequenceto form a chimeric C1qa molecule, a mouse or rat C1qb sequence is linkedto a human C1qb sequence to form a chimeric C1qb molecule, a mouse orrat C1qc sequence is linked to a human C1qc sequence to form a chimericC1qc molecule. In some embodiments, the chimeric C1q polypeptidecomprises an N-terminal stalk-stem region that is substantiallynon-human and a globular head domain that is substantially human.

In some embodiments, the nucleic acid molecule encoding a chimeric C1qpolypeptide in the genome of a genetically modified non-human animalincludes a non-human C1q nucleic acid sequence (e.g., an endogenousnon-human C1q nucleic acid sequence) and a human C1q nucleic acidsequence, wherein the human C1q nucleic acid sequence encodessubstantially the globular head domain of a human C1q polypeptide.

In some embodiments, the nucleic acid molecule encoding a chimeric C1qpolypeptide in the genome of a genetically modified non-human animalincludes a non-human (e.g., endogenous) C1q nucleic acid sequence and ahuman C1q nucleic acid sequence, wherein the non-human C1q nucleic acidsequence encodes substantially the N-terminal stalk-stem region of anon-human (e.g., endogenous) C1q polypeptide.

In some embodiments, the nucleic acid molecule encoding a chimeric C1qpolypeptide is operably linked to a 5′ regulatory element(s), such asthe promoter and/or enhancer(s), of a non-human (e.g., endogenous) C1qgene.

In some embodiments, the nucleic acid molecule encoding a chimeric C1qpolypeptide in the genome is at a locus other than an endogenous C1qlocus.

In some embodiments, the chimeric C1q nucleic acid molecule in thegenome is at an endogenous C1q locus. In some such embodiments, thechimeric C1q nucleic acid molecule in the genome can result from areplacement of a nucleotide sequence of an endogenous C1q gene at itsendogenous locus with a nucleotide sequence of a cognate human C1q gene.

In some embodiments, a contiguous genomic sequence of a non-human C1qgene at an endogenous C1q locus has been replaced with a contiguousgenomic sequence of a cognate human C1q gene to form a chimeric,humanized C1q gene.

In some embodiments, a contiguous genomic sequence of a human C1q geneinserted into an endogenous non-human C1q gene includes a portion ofexon 3 of the human C1q gene such that the resulting chimeric, humanizedC1q gene encodes a chimeric C1q polypeptide comprising a globular headdomain that is substantially human. In some embodiments, a contiguousgenomic sequence of a human C1q gene inserted into an endogenousnon-human C1q gene includes a portion of exon 3 of the human C1q genethat encodes substantially the globular head domain the human C1qpolypeptide.

In some embodiments, the genomic sequence of an endogenous C1q gene thatremains at an endogenous locus after the humanization and is operablylinked to the inserted contiguous human C1q genomic sequence, includes a3′ portion of exon 2 and a 5′ portion of exon 3, and encodessubstantially the N-terminal stalk-stem region of the endogenous C1qpolypeptide.

In circumstances where a non-human C1q polypeptide and a cognate humanC1q polypeptide share common amino acids near the junction between thestalk-stem region and the globular head domain, it may not be necessaryto insert a human C1q nucleic acid that encodes precisely the globularhead domain of the human C1q polypeptide. It is possible to insert aslightly longer or shorter nucleic acid of a human C1q gene that encodessubstantially the globular head domain of the human C1q polypeptide, inoperable linkage to a nucleic acid that encodes substantially thestalk-stem region of the non-human animal C1q polypeptide, such that thechimeric C1q polypeptide includes a globular head domain that issubstantially or fully identical to the globular head domain of thehuman C1q polypeptide, and a stalk-stem region that is substantially orfully identical to stalk-stem region of the non-human C1q polypeptide.Similarly, in circumstances where a non-human C1q polypeptide and ahuman C1q polypeptide share common amino acids near the C-terminus ofthe globular head domain, it may not be necessary to utilize a human C1qnucleic acid that encodes precisely the globular head domain of thehuman C1q polypeptide. It is possible to insert a slightly shorternucleic acid of a human C1q gene that encodes substantially (i.e.,slightly shorter than) the globular head domain of the human C1qpolypeptide, in operable linkage to a non-human nucleic acid thatencodes the remainder of amino acids at the C-terminus of the globularhead domain, such that the chimeric C1q polypeptide includes a globularhead domain that is still substantially or fully identical to theglobular head domain of the human C1q polypeptide.

In some embodiments, the human C1q nucleotide sequence included in ahumanized, chimeric C1q gene also includes the 3′ untranslated region(“UTR”) of the human C1q gene, which is the last part of exon 3 in allC1q genes (i.e., C1qa, C1qb and C1qc genes). In certain embodiments, inaddition to the 3′ UTR of a human C1q gene, an additional human genomicsequence from the human C1q gene locus can also be included. Theadditional human genomic sequence can consist of at least 10-200 bp,e.g., 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp, or more,found in the human C1q gene locus immediately downstream of the 3′ UTRof the human C1q gene. In other embodiments, the human C1q nucleotidesequence included in a humanized C1q gene does not include the 3′ UTR ofthe human C1q gene; instead, the 3′ UTR of an endogenous C1q gene isincluded and follows immediately the stop codon of the humanized C1qgene.

In some embodiments, the endogenous non-human C1q nucleic acid sequenceincluded in a humanized, chimeric C1q gene (e.g., the endogenous genomicC1q sequence remaining at an endogenous locus after humanization)includes the 5′ UTR of endogenous C1q gene (which may include exon 1 andin most cases a 5′ portion of exon 2). In some embodiments, theendogenous non-human C1q nucleotide sequence included in a humanized,chimeric C1q gene also includes a nucleotide sequence (e.g., a 5′portion of exon 2) coding for the signal peptide of the endogenous C1qpolypeptide.

In some embodiments, a non-human animal provided herein is heterozygousfor a humanized C1q gene in its genome. In other embodiments, anon-human animal provided herein is homozygous for a humanized C1q genein its genome.

In certain embodiments, a non-human animal includes multiple, i.e., twoor more, chimeric C1q genes in its genome, each at an endogenous C1qlocus or a different locus. In some embodiments, the multiple chimericC1q genes are on a contiguous nucleic acid fragment at a non-endogenousC1q locus. In some embodiments, the multiple chimeric C1q genes are eachat its endogenous C1q locus. For example, two or all three endogenousC1q genes (C1qa, C1qb and C1qc) in a non-human animal have beenhumanized using nucleotide sequences of cognate human C1q genes.

In various embodiments provided, the genetically modified non-humananimal expresses the polypeptide(s) encoded by the chimericnon-human/human C1q nucleic acid molecules. Thus, disclosed herein is agenetically modified non-human animal, wherein the non-human animalexpresses one or more chimeric non-human/human C1qa, C1qb, and/or C1qcpolypeptides. In such an aspect, the genetically modified non-humananimals can express one, two, three, four, five, or six chimericnon-human/human C1qa polypeptides, one, two, three, four, five, or sixchimeric non-human/human C1qb polypeptides, and/or one, two, three,four, five, or six chimeric non-human/human C1qc polypeptides. Invarious embodiments, the expressed chimeric C1q polypeptides arefunctional C1q polypeptides. In various embodiments, the C1qpolypeptides provided herein arrange in a typical C1q bouquet structureto form a functional C1q protein comprising 18 polypeptide chains.

In some embodiments, the non-human animal does not express a functionalendogenous C1q polypeptide (e.g., an endogenous C1qa, C1qb or C1qcpolypeptide). In some embodiments, the non-human animal does not expressa functional endogenous C1qa polypeptide, a functional endogenous C1qbpolypeptide, or a functional endogenous C1qc polypeptide. The lack ofexpression of a functional endogenous C1q polypeptide(s) can be a resultof inactivation, deletion, and/or humanization of the endogenous C1qgene(s).

In some aspects, the non-human animal expresses one or more chimericC1qa, C1qb, and/or C1qc polypeptides comprising the globular headdomains of human C1qa, C1qb, and/or C1qc polypeptides. In some aspects,the non-human animal expresses a chimeric C1qa polypeptide comprising anglobular head domain of human C1qa set forth in SEQ ID NO: 4 or afragment thereof (for example, amino acids 112-245, 122-235 or 122-222as set forth in SEQ ID NO: 4). In some aspects, the non-human animalexpresses a chimeric C1qb polypeptide comprising a globular head domainof human C1qb set forth in SEQ ID NO: 5 or a fragment thereof (forexample, amino acids 118-251, 120-251 or 125-233 as set forth in SEQ IDNO: 5). In some aspects, the non-human animal expresses a chimeric C1qcpolypeptide comprising a globular head domain of human C1qc set forth inSEQ ID NO: 6 or a fragment thereof (for example, amino acids 118-234 oramino acids 114-245 as set forth in SEQ ID NO: 6). In some aspects, thenon-human animal expresses the chimeric C1qa as set forth in SEQ ID NOs:10 or 55, C1qb as set forth in SEQ ID NOs: 11 or 56, and/or C1qcpolypeptide as set forth in SEQ ID NOs: 12 or 57.

In one aspect, it is understood and herein contemplated that thedisclosed genetically modified non-human animals comprise nucleic acidsencoding C1q polypeptides that comprise non-human amino acid sequencesand human amino acid sequences. It is further understood that all or aportion of the globular head domain of chimeric C1q is comprised ofhuman amino acid sequences. In one aspect, disclosed herein is agenetically modified non-human animal (such as, for example a rodentsuch as a mouse or rat), wherein the nucleic acid sequence encoding theglobular head of a human C1qa polypeptide or a fragment thereof encodesat least amino acids 122-222 of the human C1qa polypeptide as set forthin SEQ ID NO: 4 (for example, a nucleic acid sequence encoding aminoacids 122-235 or 112-245 of the human C1qa polypeptide as set forth inSEQ ID NO: 4). In one aspect, the genetically modified non-human animalis a rat and further comprises, operably linked to the nucleic acidsequence encoding the globular head domain or the fragment thereof ofthe human C1qa polypeptide, a nucleotide sequence encoding at leastamino acids 33-102, 30-102, 25-105, 23-107 or 23-111 of the rat C1qapolypeptide set forth in SEQ ID NO: 7, or the genetically modifiednon-human animal is a mouse and further comprises, operably linked tothe nucleic acid sequence encoding the globular head domain or thefragment thereof of the human C1qa polypeptide, a nucleotide sequenceencoding at least amino acids 33-102, 30-102, 25-105, 23-107 or 23-111of the mouse C1qa polypeptide set forth in SEQ ID NO: 1. In one aspect,the genetically modified non-human animals comprise one or more nucleicacid sequences encoding at least amino acids 23-245 of a C1qapolypeptide as set forth in SEQ ID NO: 10 or SEQ ID NO: 55. In aspecific aspect, disclosed herein are genetically modified non-humananimals (such as, for example a rodent such as a mouse or rat), whereinthe non-human animal comprises one or more nucleic acid sequencesencoding a C1qa polypeptide as set forth in SEQ ID NO: 10 or SEQ ID NO:55. In some embodiments, a genetically modified non-human animal is amouse that comprises a nucleic acid encoding a C1qa polypeptide as setforth in SEQ ID NO: 10. In some embodiments, a genetically modifiednon-human animal is a rat that comprises a nucleic acid encoding a C1qapolypeptide as set forth in SEQ ID NO: 55.

In another aspect, disclosed herein is a genetically modified non-humananimal (such as, for example a rodent such as a mouse or rat), whereinthe nucleic acid sequence encoding the globular head of a human C1qbpolypeptide or a fragment thereof encodes at least amino acids 125-233of the human C1qb polypeptide as set forth in SEQ ID NO: 5 (for example,a nucleic acid sequence encoding amino acids 120-250 or 118-251 of thehuman C1qb polypeptide).

Also disclosed herein is a genetically modified non-human animal (suchas, for example a rodent such as a mouse or rat) wherein the geneticallymodified non-human animal is a rat and comprises, operably linked to thenucleic acid sequence encoding the globular head domain or the fragmentthereof of the human C1qb polypeptide, a nucleotide sequence encoding atleast amino acids 32-105, 27-105, 27-110, 26-114, or 26-117 of the ratC1qb polypeptide set forth in SEQ ID NO: 8, or wherein the geneticallymodified non-human animal is a mouse and further comprises, operablylinked to the nucleic acid sequence encoding the globular head domain orthe fragment thereof of the human C1qb polypeptide, a nucleotidesequence encoding at least amino acids 32-105, 27-105, 27-110, 26-114,or 26-117 of the mouse C1qb polypeptide set forth in SEQ ID NO: 2.

In one aspect, the genetically modified non-human animal comprises oneor more nucleic acid sequences encoding at least amino acids 26-251 of aC1qb polypeptide as set forth in SEQ ID NO: 11 or SEQ ID NO: 56. In aspecific aspect, disclosed herein is a genetically modified non-humananimal (such as, for example a rodent such as a mouse or rat), whereinthe non-human animal comprises one or more nucleic acid sequencesencoding a C1qb polypeptide as set forth in SEQ ID NO: 11 or SEQ ID NO:56. In some embodiments, a genetically modified non-human animal is amouse that comprises a nucleic acid encoding a C1qb polypeptide as setforth in SEQ ID NO: 11. In some embodiments, a genetically modifiednon-human animal is a rat that comprises a nucleic acid encoding a C1qbpolypeptide as set forth in SEQ ID NO: 56.

In one aspect, the genetically modified non-human animal (such as, forexample a rodent such as a mouse or rat) comprises a nucleic acid,wherein the nucleic acid sequence encoding the globular head of a humanC1qc polypeptide or a fragment thereof encodes at least amino acids118-234 of the human C1qc polypeptide as set forth in SEQ ID NO: 6 (forexample, a nucleic acid sequence encoding amino acids 114-245 of thehuman C1qc polypeptide).

Also disclosed herein is a genetically modified non-human animal (suchas, for example a rodent such as a mouse or rat), wherein the non-humananimal is a rat and further comprises, operably linked to the nucleicacid sequence encoding the globular head domain or the fragment thereofof the human C1qc polypeptide, a nucleotide sequence encoding at leastamino acids 33-113, 32-115, or 32-116 of the rat C1qc polypeptide setforth in SEQ ID NO: 9, or wherein the non-human animal is a mouse andfurther comprises, operably linked to the nucleic acid sequence encodingthe globular head domain or the fragment thereof of the human C1qcpolypeptide, a nucleotide sequence encoding at least amino acids 31-111,30-113, or 30-114 of the mouse C1qc polypeptide set forth in SEQ ID NO:3.

In one aspect, the genetically modified non-human animal comprises oneor more nucleic acid sequences encoding at least amino acids 30-246 of aC1qc polypeptide as set forth in SEQ ID NO: 12 or one or more nucleicacid sequences encoding at least amino acids 32-248 of a C1qcpolypeptide as set forth in SEQ ID NO: 57. In a specific aspect,disclosed herein is a genetically modified non-human animal (such as,for example a rodent such as a mouse or rat), wherein the non-humananimal comprises one or more nucleic acid sequences encoding a C1qcpolypeptide as set forth in SEQ ID NO: 12 or SEQ ID NO: 57. In someembodiments, a genetically modified non-human animal is a mouse thatcomprises a nucleic acid encoding a C1qc polypeptide as set forth in SEQID NO: 12. In some embodiments, a genetically modified non-human animalis a rat that comprises a nucleic acid encoding a C1qb polypeptide asset forth in SEQ ID NO: 57.

In some aspect, it is beneficial for the expression of a polypeptide fora signal sequence to be present. The disclosed polypeptides anddisclosed nucleic acids encoding said polypeptides can comprise signalsequences or have signal sequences absent. Thus, in one aspect,disclosed herein is a genetically modified non-human animal (such as,for example a rodent such as a mouse or rat) wherein the non-humananimal further comprises, in operable linkage, a nucleotide sequenceencoding a rat, a mouse or a human C1qa, C1qb, and/or C1qc signalpeptide. Examples of signal peptides from rat, mouse and human C1qa,C1qb, and C1qc polypeptides are shown in FIGS. 3A-3C. For example,disclosed herein is a non-human animal comprising, in operable linkage,a nucleic acid sequence comprising a signal peptide-encoding portion ofthe rat or mouse C1qa, C1qb, and/or C1qc gene, and at least the nucleicacid sequence encoding the globular head domain or the fragment thereofof the human C1qa, C1qb, and/or C1qc polypeptide, respectively.

In one aspect, disclosed herein is a genetically modified non-humananimal (such as, for example a rodent such as a mouse or rat) comprisingin its genome a) at the endogenous C1qa locus a nucleic acid sequenceencoding a chimeric rat/human C1qa polypeptide wherein the nucleic acidsequence comprises, 5′-3′ and in operable linkage a first nucleotidesequence encoding amino acids 1-111 of a rat C1qa polypeptide of SEQ IDNO: 7 and a second nucleotide sequence encoding amino acids 112-245 of ahuman C1qa polypeptide of SEQ ID NO: 4; b) at the endogenous C1qb locusa nucleic acid sequence encoding a chimeric rat/human C1qb polypeptidewherein the nucleic acid sequence comprises, 5′-3′ and in operablelinkage a third nucleotide sequence encoding amino acids 1-117 of a ratC1qb polypeptide of SEQ ID NO: 8 and a fourth nucleotide sequenceencoding amino acids 118-251 of a human C1qb polypeptide of SEQ ID NO:5; and c) at the endogenous C1qc locus a nucleic acid sequence encodinga chimeric rat/human C1qc polypeptide wherein the nucleic acid sequencecomprises, 5′-3′ and in operable linkage a fifth nucleotide sequenceencoding amino acids 1-116 of a rat C1qc polypeptide of SEQ ID NO: 9 anda sixth nucleotide sequence encoding amino acids 114-245 of a human C1qcpolypeptide of SEQ ID NO: 6. In one embodiment, such geneticallymodified non-human animal is a rat.

Also disclosed herein is a genetically modified non-human animal (suchas, for example a rodent such as a mouse or rat) comprising in itsgenome a) at the endogenous C1qa locus a nucleic acid sequence encodinga chimeric mouse/human C1qa polypeptide wherein the nucleic acidsequence comprises, 5′-3′ and in operable linkage a first nucleotidesequence encoding amino acids 1-111 of a mouse C1qa polypeptide of SEQID NO: 1 and a second nucleotide sequence encoding amino acids 112-245of a human C1qa polypeptide of SEQ ID NO: 4; b) at the endogenous C1qblocus a nucleic acid sequence encoding a chimeric mouse/human C1qbpolypeptide wherein the nucleic acid sequence comprises, 5′-3′ and inoperable linkage a third nucleotide sequence encoding amino acids 1-117of a mouse C1qb polypeptide of SEQ ID NO: 2 and a fourth nucleotidesequence encoding amino acids 118-251 of a human C1qb polypeptide of SEQID NO: 5; and c) at the endogenous C1qc locus a nucleic acid sequenceencoding a chimeric mouse/human C1qc polypeptide wherein the nucleicacid sequence comprises, 5′-3′ and in operable linkage a fifthnucleotide sequence encoding amino acids 1-114 of a mouse C1qcpolypeptide of SEQ ID NO: 3 and a sixth nucleotide sequence encodingamino acids 114-245 of a human C1qc polypeptide of SEQ ID NO: 6. In oneembodiment, such genetically modified non-human animal is a mouse.

In one aspect disclosed herein is a genetically modified non-humananimal, wherein the non-human animal, e.g., the rat or the mouse, doesnot express a functional endogenous C1qa, C1qb, and/or C1qcpolypeptide(s).

In some embodiments, the non-human animal is a mammal. In one aspect,the non-human animal is a small mammal, e.g., of the superfamilyDipodoidea or Muroidea. In one embodiment, the genetically modifiedanimal is a rodent. In one embodiment, the rodent is selected from amouse, a rat, and a hamster. In one embodiment, the rodent is selectedfrom the superfamily Muroidea. In one embodiment, the geneticallymodified animal is from a family selected from Calomyscidae (e.g.,mouse-like hamsters), Cricetidae (e.g., hamster, New World rats andmice, voles), Muridae (true mice and rats, gerbils, spiny mice, crestedrats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasyrats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rats, bamboo rats, and zokors). In a specific embodiment,the genetically modified rodent is selected from a true mouse or rat(family Muridae), a gerbil, a spiny mouse, and a crested rat. In oneembodiment, the genetically modified mouse is from a member of thefamily Muridae. In one embodiment, the animal is a rodent. In a specificembodiment, the rodent is selected from a mouse and a rat. In oneembodiment, the non-human animal is a mouse.

In one embodiment, the non-human animal is a rodent that is a mouse of aC57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn,C57BL/10Cr, and C57BL/01a. In another embodiment, the mouse is a 129strain selected from the group consisting of a strain that is 129P1,129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 12951/SvIm), 129S2, 129S4,129S5, 12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al. (1999) Revised nomenclature for strain 129mice, Mammalian Genome 10:836, see also, Auerbach et al (2000)Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-DerivedMouse Embryonic Stem Cell Lines). In a specific embodiment, thegenetically modified mouse is a mix of an aforementioned 129 strain andan aforementioned C57BL/6 strain. In another specific embodiment, themouse is a mix of aforementioned 129 strains, or a mix of aforementionedBL/6 strains. In a specific embodiment, the 129 strain of the mix is a129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a BALBstrain, e.g., BALB/c strain. In yet another embodiment, the mouse is amix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

In some embodiments, a genetically engineered animal disclosed hereinexpresses in its serum a humanized C1q protein comprising one or morechimeric C1q polypeptides. In specific embodiments, the animal expressesin its serum a C1q protein composed of chimeric C1qa, C1qb and C1qcpolypeptides, each of which comprises a globular head domain that issubstantially human. In some embodiments, the level of a humanized C1qprotein in the serum of a genetically engineered animal is comparable tothe level of the C1q protein in a control animal without thehumanization, as detected by any of conventional assays such as WesternBlot or ELISA. By “comparable” it is meant to refer to level and valueswithin a variation of e.g., 10%, 20%, 30%, or 40%.

In some embodiments, a genetically engineered animal expressing ahumanized C1q protein in its serum displays complement activity. In someembodiments, the complement activity is detectable by a classicalhemolysis assay, as further illustrated in the examples below. Inspecific embodiments, the genetically engineered animal displaysclassical hemolytic activity in its serum at a level comparable to thelevel in a control animal without the humanization. In otherembodiments, the complement activity is detectable in an in vitrocomplement-dependent cytotoxicity (CDC) assay. In specific embodiments,the serum of a genetically engineered animal comprising a humanized C1qprotein displays CDC activity at a level comparable to that of normalhuman serum.

In a further aspect, provided herein are methods of making thegenetically modified non-human animal described herein.

In some embodiments, the method comprises modifying the genome of anon-human animal such that the modified genome comprises a nucleic acidmolecule encoding a humanized C1q polypeptide (i.e., a humanized C1qa,C1qb, and/or C1qc polypeptide).

In some embodiments, the modified genome comprises a nucleic acidencoding a chimeric C1q polypeptide, wherein the nucleic acid is locatedat a locus different from an endogenous C1q locus. In certainembodiments, a contiguous nucleic acid fragment comprising multiplenucleic acid molecules encoding different humanized C1q polypeptides islocated at a locus different from an endogenous C1q locus. For example,a contiguous nucleic acid fragment comprising a nucleic acid sequenceencoding a humanized C1qa polypeptide, a nucleic acid sequence encodinga humanized C1qb polypeptide, and a nucleic acid sequence encoding ahumanized C1qc polypeptide, is located at a locus different from theendogenous C1q locus.

In some embodiments, the modified genome comprises a nucleic acidencoding a chimeric C1q polypeptide wherein the nucleic acid is locatedat an endogenous C1q locus. In certain embodiments, a contiguous nucleicacid fragment comprising a nucleic acid sequence encoding a humanizedC1qa polypeptide, a nucleic acid sequence encoding a humanized C1qbpolypeptide, and a nucleic acid sequence encoding a humanized C1qcpolypeptide, is located at an endogenous C1q locus.

The modification to introduce a nucleic acid encoding a chimeric C1qpolypeptide into an endogenous C1q locus can, in some embodiments,result in replacement of an endogenous C1q nucleotide sequence with ahuman C1q nucleotide sequence. In one embodiment, the replacementcomprises the replacement of sequences of C1qa, C1qb, and C1qc.

Humanization may be accomplished by creating a large targeting vectorthat incorporates a genetic modification, e.g., a genetic modificationin one, two or all three C1q loci and then introducing the largetargeting vector into non-human (e.g., rodent such as mouse or rat) EScells to make a non-human animal such as a mouse, e.g., as described inExample 1, or a rat, e.g., as described in Example 2.

Thus, in one embodiment, provided herein is a large targeting vector formaking a genetically modified animal of the present disclosure. In anexemplary embodiment, the large targeting vector comprises 5′ and 3′mouse homology arms; a DNA fragment comprising the C1qa gene whichcomprises a replacement of partial sequence of mouse C1qa coding exon 3with partial sequence of human C1qa coding exon 3; a DNA fragmentcomprising the C1qb gene which comprises a replacement of partialsequence of mouse C1qb coding exon 3 with partial sequence of human C1qbcoding exon 3; a DNA fragment comprising the C1qc gene which comprises areplacement of partial sequence of mouse C1qc coding exon 3 with partialsequence of human C1qc coding exon 3; and a selection cassette. Inanother exemplary embodiment, the large targeting vector comprises 5′and 3′ rat homology arms; a DNA fragment comprising the C1qa gene whichcomprises a replacement of partial sequence of rat C1qa coding exon 3with partial sequence of human C1qa coding exon 3; a DNA fragmentcomprising the C1qb gene which comprises a replacement of partialsequence of rat C1qb coding exon 3 with partial sequence of human C1qbcoding exon 3; a DNA fragment comprising the C1qc gene which comprises areplacement of partial sequence of rat C1qc coding exon 3 with partialsequence of human C1qc coding exon 3; and a selection cassette.

In some embodiments, a large targeting vector is a genetically modifiedbacterial artificial chromosome (BAC) clone. A BAC clone carrying one ormore of non-human (e.g., rodent) C1qa, C1qb and C1qc genes can bemodified and humanized using bacterial homologous recombination andVELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela et al. (2003), High-throughput engineering of the mousegenome coupled with high-resolution expression analysis, Nature Biotech.21(6):652-659). In embodiments where the BAC clone comprises more thanone non-human C1q gene (e.g., a combination of non-human C1qa, C1qb andC1qc genes), the multiple non-human C1q genes can be sequentiallymodified through serial bacterial homologous recombination. As a result,a non-human C1q nucleotide sequence has been deleted from the originalBAC clone, and a human C1q nucleotide sequence has been inserted,resulting in a modified BAC clone carrying one or more humanized C1qgenes, flanked by 5′ and 3′ non-human homology arms.

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., bacterial cells, EScells) that have integrated the construct of interest. A number ofsuitable selection cassettes are known in the art (Neo, Hyg, Pur, CM,SPEC, etc.). In addition, a selection cassette may be flanked byrecombination sites, which allow deletion of the selection cassette upontreatment with recombinase enzymes. Commonly used recombination sitesare loxP and Frt, recognized by Cre and Flp enzymes, respectively, butothers are known in the art. A selection cassette may be locatedanywhere in the construct outside the coding region. In one embodiment,the selection cassette is inserted upstream of an inserted human C1qasequence.

The large targeting vector, such as a modified BAC clone can beintroduced into non-human (e.g., rodent) embryonic stem (ES) cells byknown techniques, e.g., electroporation. Both mouse ES cells and rat EScells have been described in the art. See, e.g., U.S. Pat. Nos.7,576,259, 7,659,442, 7,294,754, and US 2008-0078000 A1 (all of whichare incorporated herein by reference) describe mouse ES cells and theVELOCIMOUSE® method for making a genetically modified mouse; US2014/0235933 A1, US 2014/0310828 A1, Tong et al. (2010) Nature467:211-215, and Tong et al. (2011) Nat Protoc. 6(6):doi:10.1038/nprot.2011.338 (all of which are incorporated herein byreference) describe rat ES cells and methods for making a geneticallymodified rat. In some embodiments, the recipient ES cell to which amodified BAC clone is to be introduced comprises a deletion of anucleotide sequence at an endogenous C1q locus. In some embodiments, thedeletion comprises the coding region of one or more of the C1qa, C1qband C1qc genes. In specific embodiments, the deletion comprises thecoding regions of all of the C1qa, C1qb and C1qc genes; for example, adeletion that comprises the start codon of C1qa through the stop codonof C1qb. In some embodiments, the recipient ES cell is heterozygous fora deletion at an endogenous C1q locus. In other embodiments, therecipient ES cell is homozygous for a deletion at an endogenous C1qlocus.

Upon completion of gene targeting, ES cells or genetically modifiednon-human animals are screened to confirm successful incorporation ofexogenous nucleotide sequence of interest or expression of exogenouspolypeptide. Numerous techniques are known to those skilled in the art,and include (but are not limited to) Southern blotting, long PCR,quantitative PCR (e.g., real-time PCR using TAQMAN™), fluorescence insitu hybridization, Northern blotting, flow cytometry, Western analysis,immunocytochemistry, immunohistochemistry, etc. In one example,non-human animals (e.g., mice) bearing the genetic modification ofinterest can be identified by screening for loss of mouse allele and/orgain of human allele using a modification of allele assay described inValenzuela et al. (2003) High-throughput engineering of the mouse genomecoupled with high-resolution expression analysis, Nature Biotech.21(6):652-659. Other assays that identify a specific nucleotide or aminoacid sequence in the genetically modified animals are known to thoseskilled in the art. Selected ES cells are then used as donor ES cellsfor injection into a pre-morula stage embryo (e.g., 8-cell stage embryo)by using the VELOCIMOUSE® method (see, e.g., U.S. Pat. Nos. 7,576,259,7,659,442, 7,294,754, and US 2008-0078000 A1), or methods described inUS 2014/0235933 A1 and US 2014/0310828 A1. The embryo comprising thedonor ES cells is incubated until blastocyst stage and then implantedinto a surrogate mother to produce an F0 rodent. Pups bearing thehumanized C1q gene can be identified by genotyping of DNA isolated fromtail snips using loss of non-human allele and/or gain of human alleleassays. Non-human animals heterozygous for a humanized C1q gene can becrossed to generated homozygous offersprings.

In one aspect, a method for making a chimeric human/non-human C1qmolecule is provided, comprising expressing in a single cell a chimericC1q protein from a nucleotide construct as described herein. In oneembodiment, the nucleotide construct is a viral vector; in a specificembodiment, the viral vector is a lentiviral vector. In one embodiment,the cell is selected from a CHO, COS, 293, HeLa, and a retinal cellexpressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a cell that expresses a chimeric human/non-human C1qprotein is provided. In one embodiment, the cell comprises an expressionvector comprising a chimeric C1q sequence as described herein. In oneembodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinalcell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

A chimeric C1q molecule made by a non-human animal as described hereinis also provided, wherein, in one embodiment, the chimeric C1q moleculecomprises an amino acid sequence of all or substantially all of aglobular head domain of a human C1qa, C1qb, and/or C1qc polypeptides,and at least stem and/or stalk domains from a non-human C1q protein,e.g., mouse C1q protein.

In addition to a genetically engineered non-human animal, a non-humanembryo (e.g., a rodent, e.g., a mouse or a rat embryo) is also provided,wherein the embryo comprises a donor ES cell that is made as disclosedhereinabove or is derived from a non-human animal (e.g., a rodent, e.g.,a mouse or a rat) as described herein. In one aspect, the embryocomprises an ES donor cell that comprises a chimeric C1q gene, and hostembryo cells.

Also provided is a tissue, wherein the tissue is derived from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein, and expresses a chimeric C1q protein. In some embodiments, atissue is selected from blood, plasma, serum, bone marrow, spleen, lymphnodes, brain, and a combination thereof.

In addition, a non-human cell isolated from a non-human animal asdescribed herein is provided. In one embodiment, the cell is an ES cell.In one embodiment, the cell is a dendritic cell.

F. Rodent Model for Testing Human Therapies

C1q molecules are being studied as targets for bispecific agents, e.g.,bispecific antibodies, with one arm binding human C1q and anotherbinding an antigen of interest.

During preclinical drug development stage, candidate agents aretypically studied based on their efficacy, toxicity, and otherpharmacokinetic and pharmacodynamics properties. Candidate agents, suchas antibodies, typically target a human antigen—as the end goal ofinvestigation is to develop a human therapy. Many preclinical studiesare conducted in large animals such as primates as their physiology anddrug metabolism are most similar to humans. To conduct effectivepreclinical investigations relating to efficacy, toxicity, and otherparameters of a drug candidate, first, the drug candidate must bedetermined to recognize primate C1q molecule.

However, a separate factor complicating development of anti-C1q therapyis that large primates such as chimpanzees are endangered and in manycountries studies in chimpanzees are prohibited; while studies in otherprimates, e.g., cynomolgus monkeys (Macaca fascicularis), may raiseethical concerns. Thus, any preliminary data on a specific therapeuticcandidate that can be obtained in a smaller animal model, such as arodent, e.g., a mouse, can be helpful in determining further progress ofpreclinical investigations in large primates.

The most useful small animal model to conduct preliminary studies is anon-human animal, e.g., a rodent, that expresses a human or humanizedC1q protein, and allows the testing of anti-C1q drug candidates thatalso target, for example a tumor antigen, viral antigen, or bacterialantigen (such, as for example, a Staphylococcus antigen).

Accordingly, in some aspects, provided herein is a rodent model (suchas, for example, a mouse or rat model) for testing C1q-targeted(“anti-C1q”) therapeutic agents. In some embodiments, provided herein isa rodent model (such as, for example, a mouse or rat model) for testinganti-C1q antigen-binding proteins. In some embodiments, provided hereinis a rodent model (such as, for example, a mouse or rat model) fortesting anti-C1q antibodies. In some such embodiments, provided is arodent model for testing anti-C1q multi-specific, e.g. bispecific,antigen-binding proteins or anti-C1q bispecific antibodies. As such, ananti-C1q multi-specific antigen-binding protein, e.g. an anti-C1qbispecific antigen-binding protein, targets or specifically binds saidhumanized C1q polypeptide or humanized C1q complex and at least oneother antigen of interest. In various aspects, the rodent model fortesting anti-C1q bispecific antigen-binding proteins wherein theantigen-binding protein is capable of binding both a humanized C1qcomplex (with one or more C1qa, C1qb, and/or C1qc polypeptides of thecomplex being humanized) and the antigen of interest comprises a nucleicacid sequence encoding a humanized C1q complex, wherein the humanizedC1q polypeptide is selected from the group consisting of C1qa, C1qb,C1qc, and/or a combination thereof, and a cell expressing or comprisingthe antigen of interest. In one embodiment, the rodent comprises adendritic cell expressing said humanized C1q protein(s).

The term “germline” in reference to an immunoglobulin nucleic acidsequence includes a nucleic acid sequence that can be passed to progeny.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The term “antigen-binding protein” as used herein includes antibodiesand various naturally produced and engineered molecules capable ofbinding the antigen of interest. Such include, e.g., domain-specificantibodies, single domain antibodies (e.g., derived from camelids andfish, etc.), domain-deleted antibodies, chimeric antibodies, CDR-graftedantibodies, diabodies, triabodies, tetrabodies, minibodies, nanabodies(e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), shark variable IgNAR domains, T cellreceptor molecules and molecules comprising T cell receptor variabledomains and fragments thereof, etc. Antigen-binding protein may alsoinclude antigen-binding fragments such as, e.g., (i) Fab fragments; (ii)F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v)single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region of an antibody (e.g., an isolated complementaritydetermining region (CDR) such as a CDR3 peptide), etc.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable domain and a heavy chain constantregion (C_(H)). The heavy chain constant region comprises three domains,C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chainvariable domain and a light chain constant region (C_(L)). The heavychain and light chain variable domains can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each heavy and light chain variable domaincomprises three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 andHCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).

As used herein, “an antibody that binds C1q” or an “anti-C1q antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single C1q subunit (e.g., C1qa, C1qb, and/orC1qc), as well as antibodies and antigen-binding fragments thereof thatspecifically recognize a dimeric complex of two C1q subunits (e.g.,C1qa/C1qb, and C1qc/C1qc dimers) as well as trimers of dimers. Theantibodies and antigen-binding fragments can also bind soluble C1qand/or IgM or IgG bound C1q.

The term “high affinity” antibody or antigen-binding protein refers toan antibody that has a K_(D) with respect to its target epitope about of10⁻⁹ M or lower (e.g., about 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about1×10⁻¹² M).

The phrase “bispecific antibody” or “bispecific antigen-binding protein”includes an antibody or antigen-binding protein capable of selectivelybinding two epitopes. Bispecific antibodies generally comprise two arms,each binding a different epitope (e.g., two heavy chains with differentspecificities)—either on two different molecules (e.g., differentepitopes on two different immunogens) or on the same molecule (e.g.,different epitopes on the same immunogen). If a bispecific antibody orantigen-binding protein is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst antibody arm for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first antibody arm for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Exemplary bispecific antibodies include those with a firstantibody arm specific for C1q, and a second antibody arm specific for anantigen of interest (e.g., an antigen of an infectious agent).Bispecific antibodies can be made, for example, by combining heavychains that recognize different epitopes of the same immunogen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same immunogen can be fused tonucleic acid sequences encoding the same or different heavy chainconstant regions, and such sequences can be expressed in a cell thatexpresses an immunoglobulin light chain. A typical bispecific antibodyhas two heavy chains each having three heavy chain CDRs, followed by(N-terminal to C-terminal) a C_(H)1 domain, a hinge, a C_(H)2 domain,and a C_(H)3 domain, and an immunoglobulin light chain that either doesnot confer epitope-binding specificity but that can associate with eachheavy chain, or that can associate with each heavy chain and that canbind one or more of the epitopes bound by the heavy chainepitope-binding regions, or that can associate with each heavy chain andenable binding of one or both of the heavy chains to one or bothepitopes. Similarly, the phrase “multispecific antibody” includes anantibody capable of selectively binding multiple epitopes (e.g., two,three, four epitopes).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule. A CDR can beencoded by, for example, a germline sequence or a rearranged orunrearranged sequence, and, for example, by a naive or a mature B cell.A CDR can be somatically mutated (e.g., vary from a sequence encoded inan animal's germline), humanized, and/or modified with amino acidsubstitutions, additions, or deletions. In some circumstances (e.g., fora CDR3), CDRs can be encoded by two or more sequences (e.g., germlinesequences) that are not contiguous (e.g., in an unrearranged nucleicacid sequence) but are contiguous in a B cell nucleic acid sequence,e.g., as the result of splicing or connecting the sequences (e.g., V-D-Jrecombination to form a heavy chain CDR3).

The phrase “functional fragment” includes fragments of antigen-bindingproteins such as antibodies that can be expressed, secreted, andspecifically bind to an epitope with a K_(D) in the micromolar,nanomolar, or picomolar range. Specific recognition includes having aK_(D) that is at least in the micromolar range, the nanomolar range, orthe picomolar range.

The phrase “heavy chain,” as in “immunoglobulin heavy chain”, includesan immunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. A heavy chain variable domain is encoded by a variable region genesequence, which generally comprises V_(H), D_(H), and J_(H) segmentsderived from a repertoire of V_(H), D_(H), and J_(H) segments present inthe germline. Sequences, locations and nomenclature for V, D, and Jheavy chain segments for various organisms can be found on the websitefor the International Immunogenetics Information System (IMGT database).

The phrase “light chain”, as in “immunoglobulin light chain”, includesan immunoglobulin light chain sequence from any organism, and unlessotherwise specified includes human kappa and lambda light chains and aVpreB, as well as surrogate light chains. Light chain variable domainstypically include three light chain CDRs and four framework (FR)regions, unless otherwise specified. Generally, a full-length lightchain includes, from amino terminus to carboxyl terminus, a variabledomain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chainconstant region. A light chain variable domain is encoded by a lightchain variable region gene sequence, which generally comprises V_(L) andJ_(L) segments, derived from a repertoire of V and J segments present inthe germline. Sequences, locations and nomenclature for V and J lightchain segments for various organisms can be found on the website for theInternational Immunogenetics Information System (IMGT database). Lightchains include those, e.g., that do not selectively bind any epitopesrecognized by antigen-binding protein (e.g., antibody) in which theyappear. Light chains also include those that bind and recognize, orassist the heavy chain with binding and recognizing, one or moreepitopes selectively bound by the antigen-binding protein (e.g., anantibody) in which they appear.

In various embodiments, the antigen-binding protein binds both C1q andan antigen of interest.

In another embodiment, the rodent model is used to determine if acandidate bispecific antigen-binding protein is capable of blocking oraffecting an antigen of interest which is an infectious diseaseassociated antigen. In one embodiment, the rodent is infected with aninfectious agent. In one embodiment, the infectious disease associatedantigen is a viral antigen.

In another embodiment, wherein the antigen of interest is an infectiousdisease associated antigen, the antigen of interest is a bacterialantigen. In some aspects, the bacterial antigen is a Staphylococcusantigen.

In some aspects, the C1q-based bispecific antigen binding protein is ahuman C1q based antigen binding protein. In one embodiment, the antigenbinding protein is an antibody, e.g., a human antibody, or anantigen-binding fragment thereof.

In some embodiments, the testing of a bispecific antibody with one armtargeting human C1q and another arm targeting an infectious diseaseassociated antigen (such as S. aureus), is done in vivo using agenetically engineered animal disclosed herein that expresses ahumanized C1q having a human or substantially human globular head domainin each of its C1qa, C1qb and C1qc polypeptide chains. The animal can beinfected with the infectious disease associated antigen (such as S.aureus), and the bispecific antibody can be evaluated in such animal,e.g., in its ability to reduce bacterial burden and/or improve survival.

G. Use of Genetically Modified Non-Human Animals

Further disclosed are various methods of using the genetically modifiednon-human animals described herein.

In one embodiment, provided herein is a method of screening therapeuticdrug candidates that target an antigen of interest comprising (a)providing or receiving a genetically modified rodent (such as a mouse orrat) comprising at its endogenous rodent C1q locus a nucleic acidsequence encoding a chimeric, humanized C1qa polypeptide, C1qbpolypeptide and/or C1qc polypeptide and/or any combination thereof, (b)introducing into said genetically modified rodent an antigen ofinterest, (c) contacting said rodent with a drug candidate of interest,wherein the drug candidate is directed against the human C1q and theantigen of interest, and (d) assaying if the drug candidate isefficacious in preventing, reducing or eliminating cells or virusescharacterized by the presence or expression of the antigen of interest.In various embodiments, the rodent expresses a functional humanized C1qcomplex. In one embodiment of the method, the genetically modifiedrodent comprises at the endogenous rodent C1q locus a nucleic acidsequence encoding a chimeric C1qa polypeptide comprising a globular headdomain that is substantially human, a chimeric C1qb polypeptidecomprising a globular head domain that is substantially human, and achimeric C1qc polypeptide comprising a globular head domain that issubstantially human. In one embodiment of the method described herein,the rodent does not comprise a nucleic acid sequence encoding afunctional globular head domain of the corresponding rodent protein.

In various embodiments of the method described herein, introduction ofthe antigen of interest into the genetically modified rodent describedherein may be accomplished by any methods known to those skilled in theart, which may include, without limitation, transgenesis, injection,infection, tissue or cell transplantation. As such, introduction may beachieved by expressing in the rodent the antigen of interest, which cancomprise genetically modifying said rodent to express the antigen ofinterest. Alternatively, introduction may comprise introduction intosaid rodent a cell expressing the antigen of interest, e.g., as in cellor tissue transplantation. Introduction may also comprise infecting saidrodent with the antigen of interest, e.g., as in bacterial or viralinfection. In one embodiment, the antigen of interest may be a humanantigen of interest. In another embodiment, it may be a bacterial or aviral antigen of interest. The antigen of interest may be atumor-associated antigen or an infectious disease associated antigen,e.g., a bacterial or a viral antigen, as described in detail above.

In another embodiment, provided herein is a method of assessing orscreening therapeutic drug candidates that target an antigen of interestcomprising mixing a cell or virus expressing the antigen of interestwith (i) a drug candidate of interest, wherein the drug candidate isdirected against the human C1q and the antigen of interest, and (ii) ablood sample (e.g., a whole blood sample) of a genetically modifiedrodent described herein, and (b) assaying to determine whether the drugcandidate is efficacious in reducing or eliminating the cell or viruscharacterized by the presence or expression of the antigen of interest.The determination can be made based on measuring, e.g., percentagesurvival of the cell or virus where a drug candidate is used as comparedto a control drug or no drug at all. The antigen of interest may be atumor-associated antigen or an infectious disease associated antigen,e.g., a bacterial or a viral antigen, as described in detail above. Insome embodiments, the antigen of interest is a bacterial antigen such asa Staphylococcus antigen. In some embodiments, the cell is a bacterialcell such as a Staphylococcus cell.

In various embodiments of the methods of screening a therapeutic drugcandidate, the drug candidate may be an antigen-binding protein, e.g.,an antibody, e.g., a bispecific antibody. In various aspects, such drugcandidate is capable of binding both human C1q and the antigen ofinterest. The antigen of interest may be a human antigen. The antigen ofinterest may also be a primate, e.g., a monkey, antigen. Thus, the drugcandidate used for screening may be capable of binding both a humanantigen and a corresponding primate antigen, in addition to bindinghuman C1q. The drug candidate may also be capable of binding primate,e.g., monkey, C1q. Thus, the drug candidate may be capable of bindingboth human and primate, e.g., monkey, C1q; and also, in one embodiment,be capable of binding a human antigen of interest. In anotherembodiment, the antigen of interest may be a bacterial or a viralantigen, and the drug candidate may be capable of binding both the humanand primate, e.g., monkey, C1q and the antigen of interest (e.g., aviral or bacterial antigen).

In various embodiments of the methods described herein, the therapeuticcandidate is capable of reducing, eliminating, or preventing a disease.In one embodiment, the disease is a tumor, and the therapeutic candidateis capable of reducing, eliminating, or preventing tumor growth ascompared to an agent that does not target the antigen of interest. Insuch an embodiment of the method, determination whether the drugcandidate is efficacious in preventing, reducing or eliminating cellscharacterized by the presence or expression of the antigen of interestcan be performed using a tumor volume assay, a tumor cell killing assay,induction of apoptotic markers in tumors, reduction in blood vesselgrowth in tumors, infiltration of immune cells into tumors, etc. Inanother embodiment, the disease is an infectious disease, and atherapeutic candidate is capable reducing, eliminating, or preventing abacterial or a viral infection as compared to an agent that does nottarget the antigen of interest. In such an embodiment of the method,determination whether the drug candidate is efficacious in preventing,reducing or eliminating cells or viruses characterized by the presenceor expression of the antigen of interest can be performed using ameasure of bacterial or viral titers, induction of apoptotic markers ininfected cells, etc., or by measuring survival of bacterial cells orviruses using a blood sample (e.g., a whole blood sample).

In addition to evaluating bispecific antibodies with one arm targeting ahumanized C1q protein and the other arm targeting an antigen ofinterest, the genetically engineered non-human animals expressing ahumanized C1q protein disclosed herein are useful for evaluating theeffects of other antibodies, e.g., monospecific antibodies having ahuman Fc region (such as a human antibody). The binding of a humanizedC1q to the human Fc region of an antibody can activate classicalcomplement pathway, which leads to complement-dependent cytotoxicity(CDC). There had been a difficulty in assessing whether an antibody hasan effect on the complement system of a recipient, or whether or howmuch of the efficacy of a therapeutic antibody is attributable to theaction of the complement system. The genetically engineered non-humananimals expressing a humanized C1q disclosed herein will permitassessment of whether an antibody having a human Fc region (e.g., ahuman antibody) will be capable of activating classical complementpathway, and the results of such assessment will more accurately reflectwhether such antibody will activate classical complement pathway whengiven to human patients.

Therefore, in a further aspect, disclosed herein is a method ofassessing whether an antibody comprising a human Fc region can activateclassical complement pathway by utilizing a genetically engineerednon-human animal (e.g., a rodent such as a mouse or rat) expressing ahumanized C1q protein disclosed herein.

In some embodiments, the method utilizes a cell expressing an antigen ofinterest on the cell surface, a candidate antibody comprising a human Fcregion and directed to the antigen of interest, and a serum sample froma genetically engineered non-human animal expressing a humanized C1qprotein, and is designed to evaluate in vitro complement-dependentcytotoxicity of the cell expressing the antigen of interest. In specificembodiments, the cell is first mixed with the candidate antibody toallow the antibody to bind to the antigen of interest expressed on thecell surface; then a serum sample is added to the cell-antibody mixtureto permit binding of the C1q proteins in the serum sample to antibodiesbound to the antigen of interest on the cell. Cytotoxicity (i.e.,killing of said cell) can then be measured using reagents readilyavailable, including those from commercial sources (e.g., CytoTox-Glo′reagent from Promega), using flow cytometry methods or release ofpre-loaded radioisotopes from target cells. Cytotoxicity using a serumsample from a humanized non-human animal expressing a humanized C1qprotein can be compared with a serum sample from a control non-humananimal without the humanization (negative control), and with a humanserum sample (positive control).

In some embodiments, cells suitable for use in this method include Rajicells, Ramos cells, Daudi cells, HEK293 cells, and A431 cells. In someembodiments, Raji cells, Ramos cells, or Daudi cells are used in thepresent methods. The cells can naturally express an antigen of intereston the cell surface, or can be modified to recombinantly express anantigen of interest on the cell surface.

In some embodiments, the candidate antibody is a human antibody directedto a tumor antigen, a bacterial or viral antigen, etc. Examples ofcandidate antibodies include, e.g., anti-CD20, etc.

In some embodiments, the methods of assessing whether an antibodycomprising a human Fc region can activate classical complement pathwayis performed in vivo by comparing the effects of a candidate antibody ina C1q humanized animal with a C1q knockout animal. To rule out that theeffect is due to ADCC (as opposed to CDC), NK cells, neutrophils andmacrophages in the animals could be depleted, leaving the complementsystem intact.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1. Generation of Humanized C1q Mouse

Mouse genomic sequence of C1q genes can be found under NCBI AccessionNumber NC_000070.6, and C1q loci are located at mouse chromosome 4D3(Reference GRCm38.p4 C57BL/6J). Human genomic sequence of C1q genes canbe found under NCBI Accession Numbers NG 007281.1, NG 007283.1 and NG007565.1, and C1q loci are located at human chromosome 1p36.1. Someexamples of genomic and amino acid sequences for human and mouse C1q arelisted below in Tables 1 and 2. Predicted signal peptide in the listedSEQ ID NOs boundaries are indicated. The signal peptide boundaries arealso boxed in FIGS. 3A-3C.

TABLE 1 GeneBank Accession Numbers for Mouse C1q sequences SignalGenomic Sequence Sequence ID No. peptide NCBI Accession Protein NCBI(Immature (amino Protein/Gene Name Number Accession Number Protein)acids) Mouse C1qa NC_000070.6 NP_031598.2 1 1-22 Mouse C1qb NC_000070.6NP_033907.1 2 1-25 Mouse C1qc NC_000070.6 NP_031600.2 3 1-29

TABLE 2 GeneBank Accession Numbers for Human C1q Sequences GenomicSequence Sequence ID No. Protein/Gene NCBI Accession Protein NCBI(Immature Signal Name Number Accession Number Protein) peptide HumanC1QA NG_007282.1 NP_001334394.1 4 1-22 Human C1QB NG_007283.1NP_000482.3 5 1-27 Human C1QC NG_007565.1 NP_001334548.1 6 1-28

Briefly, to generate chimeric C1q mice, the mouse C1q locus washumanized by construction of a unique targeting vector from human andmouse bacterial artificial chromosomes (BAC) DNA using VELOCIGENE®technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al.(2003) High-throughput engineering of the mouse genome couple withhigh-resolution expression analysis. Nat. Biotech. 21(6): 652-659, bothincorporated herein by reference) using the mouse BAC library Mouse BACES release 2 (Incyte Genomics, 129/SvJ in pBeloBAC11). DNA from mouseBAC clone 302p21 was modified to replace genomic DNA encoding portionsof mouse C1qa, C1qb, and C1qc (mouse C1q genes are located in closeproximity to one another on the reverse strand of mouse chromosome 4)with corresponding portions of human C1qa, C1qb, and C1qc, respectively(human C1q genes are located in close proximity to one another on theforward strand of human chromosome 1).

The mouse C1q BAC was modified by introduction of human C1q sequences,to generate a BAC comprising humanized C1qa, C1qb, and C1qc genes. Thesequences encoding substantially the mouse C1qa, C1qb, and C1qc globulardomains were replaced, respectively, with the corresponding sequencesencoding substantially the human C1qa, C1qb, and C1qc globular domains.Alignments of human and mouse C1q (and rat) protein sequences aredepicted in FIGS. 3A, B, and C, where the boundaries for globular headsare as indicated, and the boundaries of mouse/human genes are indicatedwith arrows. The amino acid sequences of the humanized mouse C1qa, C1qb,and C1qc proteins are set forth in SEQ ID NOs: 10, 11, and 12,respectively, and are listed in Table 3 below, with mouse sequencesitalicized.

TABLE 3 Amino Acid Sequences of the Chimeric Mouse/Human C1q proteinsSEQ ID Protein Sequence NO: C1qaMETSQGWLVACVLTMTLVWTVAEDVCRAPNGKDGAPGNPG 10RPGRPGLKGERGEPGAAGIRTGIRGFKGDPGESGPPGKPGNVGLPGPTGPLGDSGPQGLKGVKGNPGNIRD QPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA* C1qbMKTQWGEVWTHLLLLLLGFLHVSWAQSSCTGPPGIPGIPGV 11PGVPGSDGQPGTPGIKGEKGLPGLAGDLGEFGEKGDPGIPGTPGKVGPKGPVGPKGTPGPSGPRGPKGDSGDYGATQKIAFSATRTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA* C1qcMVVGPSCQPPCGLCLLLLFLLALPLRSQASAGCYGIPGMPGMP 12GAPGKDGHDGLQGPKGEPGIPAVPGTRGPKGQKGEPGMPGHRGKNGPRGTSGLPGDPGPRGPPGEPGVEGR YKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD*

Example 1.1. Generation of C1q Knock Out Mouse

In detail, first, a mouse C1q locus comprising all three C1q genes wasmodified to delete the 17.6 kB nucleotide sequence comprising genesencoding mouse C1q (see FIG. 1A). A targeting vector comprising LacZ-neocassette, 20 Kb 5′ homology arm and 55 kb 3′ homology arm was introducedinto mouse BAC 302p21 by bacterial homologous recombination such that17.6 Kb of nucleotide sequence comprising all three mouse C1q genes fromC1qa ATG to C1qb stop codon was deleted. The deletion spanned C1qa exon2 just after the start ATG through C1qb exon 3 past the stop codonincluding 19 bp into the C1qb 3′ UTR. The cassette was inserted suchthat the LacZ coding sequence was in frame with the C1qa ATG codon. LacZcoding sequence is followed by an SV40 polyadenylation site, then afloxed neomycin resistance cassette under control of the mousephosphoglycerate kinase 1 (Pgk1) promoter with Pgk1 polyadenylationsignal. The resultant vector was used to electroporate mouse ES cells tocreate modified ES cells for generating a mouse that lacked theendogenous C1q locus. The sequences of the various junctions in thedeleted locus are labeled in the second schematic diagram in FIG. 1C,with corresponding nucleic acid sequences listed in Table 4 below. Thejunctions shown in Tables in this example only list short nucleotidesequences, but direct one skilled in the art to the location where thesequences were inserted into the mouse genome.

TABLE 4 Junction Sequences of the mouse C1q Knock Out Locus JunctionDescription Sequence SEQ ID NO Mouse CATACCCAGTGTCCCTGTGTGTCTCTGTAGGGACA13 C1qa/KpnI/lacZ CCATG/GGTACC/GATTTAAATGATCCAGTGGTC Pgk1GCAGCCCCTAG/ATAACTTCGTATAATGTATGCT 14 polyA/loxP/XhoI/ATACGAAGTTAT/CCTAGG/CTATCCAACACCATCT Mouse C1qb TCCTGC

ES cells containing deletion of mouse C1q sequences were identified by aquantitative TAQMAN™ assay (see, e.g., Lie and Petropoulos, 1998. Curr.Opin. Biotechnology 9:43-48, incorporated herein by reference),modification of allele assay (MOA). Specific primer sets and probes weredesigned for detecting insertion of the cassette sequences(gain-of-allele, GOA) and deletion of mouse sequences (loss-of-allele,LOA). Table 5 identifies the names and locations of each ofprimers/probe sets used in the quantitative PCR assays.

TABLE 5Primer and Probes Used in a MOA Assay to Confirm Deletion of mouse C1q locusLoss of Allele (LOA) or Gain SEQ Description Sequence of Allele (GOA)ID NO 598TU Probe TCCCGCACCATCCTGGAGGCAAT LOA (mouse 15 FTAAGCGTTCTCTCCGGCTGG C1qa) 16 R CGCTTCTCAGGACCCCTAAAC 17 LacZ probeCGATACTGTCGTCGTCCCCTCAAACTG GOA 18 F GGAGTGCGATCTTCCTGAGG 19 RCGCATCGTAACCGTGCATC 20 Neo probe TGGGCACAACAGACAATCGGCTG GOA 21 FGGTGGAGAGGCTATTCGGC 22 R GAACACGGCGGCATCAG 23 587mTD ProbeAGGACCATCAACAGCCCCTTGCGAC LOA (mouse 24 F GAAAGTCGCCTTCTCTGCCC C1qb) 25R CGAAGCGAATGACCTGGTTC 26

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donorES cell) independently bearing a mouse C1q deletion were identified bygenotyping using a modification of allele assay (see above) that detectsthe absence of mouse C1q gene sequences. Modified mouse ES cellscomprising the deleted mouse C1q locus (mouse C1q KO HET ES cells) wereused for humanized C1q construction as described below.

The selection cassette used in this method may be removed by methodsknown by the skilled artisan. For example, ES cells bearing the C1qknock out locus may be transfected with a construct that expresses Crein order to remove the foxed cassette. The selection cassette mayoptionally be removed by breeding to mice that express Cre recombinase.Optionally, the selection cassette is retained in the mice.

Example 1.2. Generation of Humanized C1q Mouse

To generate humanized mouse C1q, donor plasmids were generated byIn-Fusion HD Cloning Kits' (Clontech) using sequences for C1q globularhead domains (the same sequences as used for the humanized C1q rat inthe Example below) and overlapping mouse sequences. For C1qbhumanization construct, a loxP-Ub-Hyg selection cassette was inserted byrestriction digest downstream of the human C1qb polyA sequence. For bothC1qa and C1qc constructs, a spectinomycin (Spec) selection cassette wasinserted by restriction digest downstream of each C1qa and C1qc polyAsequences. The resultant constructs were introduced sequentially intothe mouse C1q BAC (302p21) through bacterial homologous recombinationfollowed by selection and/or digestion steps to remove the selectioncassettes, as demonstrated in FIG. 1B. In this particular embodiment,the chimeric nucleic acid sequence for C1qb was introduced first (1 inthe figure), followed by introduction of chimeric C1qc DNA (2 in thefigure), and then chimeric C1qa DNA (3 in the figure).

The large targeting vector containing all three chimeric C1q genes waselectroporated into mouse C1q KO HET ES cells as depicted in FIG. 1C,and successful integration was confirmed by a TAQMAN® real-timePCT-based modification of allele (MOA) assay described above. Primersand probes used for the MOA assay, and their locations, are described inTable 6.

TABLE 6Primer and Probes Used in MOA Assay to Confirm Presence of ChimericMouse/Human C1q genes Loss of Allele (LOA) or Gain of Allele SEQ IDDescription Sequence (GOA) NO 1565ma1 TGACAAGGTCCTCACCAACCAGGAGAGLOA (mouse 27 F CGCTTGGCAACGTGGTTAT C1qa) 28 R CCCGTGTGGTTCTGGTATGG 291565mb1 TATGAGCCACGCAACGGCAAGTTCA LOA (mouse 30 F TCACCAACGCGAACGAGAAC1qb) 31 R GGCCAGGCACCTTGCA 32 1565mc5 CCCATCCTCACTCAGACCTCTTCCTCCALOA (mouse 33 F CACCTCGCTCCCTCTGCTT C1qc) 34 R CAGGAACCAGGGTGGACTTC 351565ha1 CAACGTGGTCATCTTCGACACGGTCA GOA (human 36 F CGGAACCCCCCAATGGC1qa) 37 R TGGTTCTGGTACGGTTCTTCCT 38 1565hb2 ACCATCAACGTCCCCCTGCGCGOA (human 39 F AATCGCCTTCTCTGCCACAA C1qb) 40 R GTGGTCGAAGCGGATGGT 411565hc4 CACCTGCAAAGTCCCCGGCCTC GOA (human 42 F TGACACGAGCACTGGCAAGTC1qc) 43 R CGACGCGTGGTAGACAAAGTAG 44 Hyg ACGAGCGGGTTCGGCCCATTC LOA 45 FTGCGGCCGATCTTAGCC (hygromycin 46 R TTGACCGATTCCTTGCGG resistance 47deletion after introduction of Cre)

Junction sequences between various genetically engineered components atthe chimeric locus are depicted in Table 7 below, and are indicated onthe bottom schematic diagram in FIG. 1C. The junctions shown in Tablesin this Example only list short nucleotide sequences, but direct oneskilled in the art to the location where the sequences were insertedinto the mouse genome.

TABLE 7 Junction Sequences of the Chimeric Human/Mouse C1q LocusJunction SEQ ID Description Sequence NO Mouse C1qaCGGCCCCCAAGGACTGAAGGGCGTGAAAGGCAATCCA 48 exon 3/humanGGCAATATCAGGGAC/CAGCCGAGGCCAGCCTTCTCCG C1QA (globularCCATTCGGCGGAACCCCCCAATGGGGGGC domain) Human C1QATTGAGAGGGAGGCCTAAGAATAATAACAATCCAGTG 49 3′ UTR/AsiSICTTAAGAGTCAGGC/GCGATCGC/TGATGCACGCCTTT site/3′ of mouseAATCCCAGCACTTGGGAGGCAGAGACAGGTGA C1qa (non- coding) Mouse C1qcGCCAGGGGACCCAGGCCCCAGGGGGCCTCCGGGGG 50 exon 3/humanAGCCAGGTGTGGAGGGCCGA/TACAAGCAGAAATTC C1QC (globularCAGTCAGTGTTCACGGTCACTCGGCAGACCCACCA domain) Human C1QC 3′TGAATTTCGGATCTTCAACTTTGCATCAGCCATAGCT 51 UTR/3′ ofGGGCTCTGGACTC/TACCTAACTATAACGGTCCTAAGGT mouse C1qcAGCGAAAGGGGGATGATTTGGAGT (non-coding) Mouse C1qbTCCAGGCCCCTCTGGACCCCGCGGTCCCAAAGGCGATT 52 exon 3/ humanCTGGGGACTACGGGGCTACA/CAGAAAATCGCCTTCTC C1QB (globularTGCCACAAGAACCATCAACGTCCCCCTGCGCCGGG domain) Human C1QBGCACCTGGCACACCAGAAGTGCCATGCTCAGAAATG 53 3′UTR/ loxp-TTGGTTACATGAATGAAT/GCGGCCGCACCGGTATAA Ub-Hyg regionCTTCGTATAATGTATGCTATACGAAGTTA loxp-Ub-HygCCGGCGCGCCATAACTTCGTATAATGTATGCTATACG 54 region/ 3′ ofAAGTTATGTCGAC/GAATGTTCATAGGCTGGGGAGATG mouse C1qbGCTCAGTCAGTAAAGTACTTAGCTTGC (non-coding)

Targeted ES cells described above are used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® methoddescribed above. VELOCIMICE® independently bearing a humanized C1q genesare identified by genotyping using a modification of allele assay (seeabove) that detects the presence of the unique human C1q gene sequences.Mice comprising a heterozygous modification of the C1q genes are bred tohomozygousity.

To generate ES cells without the hygromycin resistance cassette, aplasmid containing cre recombinase coding sequence was electroporatedinto mouse C1q KO HET ES cells and resulting ES cell clones werescreened for loss of the hygromycin resistance cassette by TaqMan assayusing primers and probe depicted in Table 6. Selected clones were thenmicroinjected into 8-cell stage mouse embryos as described above andresulting mice were bred to homozygosity. The resulting expressedchimeric proteins are shown above in Table 3 and the Sequence Listing.

Example 2. Generation of Humanized C1q Rat

Rat genomic sequence of C1q genes can be found under NCBI AccessionNumber NC_005104.4, and C1q loci are located at Rat chromosome 5(Reference Rnor_6.0 Primary Assembly). Human genomic sequence of C1qgenes can be found under NCBI Accession Numbers NG 007283.1, NG 007282.1and NG 007565.1, and C1q loci are located at human chromosome 1p36.1.Some examples of genomic and amino acid sequences for rat and human C1qare listed in Tables 8 and 2, respectively. Predicted signal peptideboundaries in the listed SEQ ID NOs are indicated. The signal peptideboundaries are also boxed in FIGS. 3A-3C.

TABLE 8 GeneBank Accession Numbers for Rat C1q sequences GenomicSequence Sequence ID No. Protein/Gene NCBI Accession Protein NCBI(Immature Signal Name Number Accession Number protein) peptide rat C1qaNC_005104.4 NP_001008515.1 7 1-22 rat C1qb NC_005104.4 NP_062135.1 81-25 rat C1qc NC_005104.4 NP_001008524.1 9 1-31

To generate chimeric C1q rat, briefly, the rat C1q locus was humanizedby construction of a unique targeting vector from synthesized humansequences and rat bacterial artificial chromosomes (BACs) DNA using therat BAC library generated for Regeneron by LUCIGEN® from rat Dark AgoutiES cells, and the rat targeting technology described in US 2014/0310828,incorporated herein in its entirety by reference. The BAC sequences wereconfirmed and updated based on the data from Next Generation Sequencing.DNA from rat BAC (rat C1q BAC, LUCIGEN®) was modified to replace genomicDNA encoding portions of rat C1qa, C1qb, and C1qc (rat C1q genes arelocated in close proximity to one another on the reverse strand of ratchromosome 5) with corresponding portions of human C1qa, C1qb, and C1qc,respectively (human C1q genes are located in close proximity to oneanother on the forward strand of human chromosome 1).

The rat C1q BAC generated in-house and described above was modified byintroduction of human C1q sequences, to generate a vector comprisinghumanized C1qa, C1qb, and C1qc genes. The sequences encoding themajority of rat C1qa, C1qb, and C1qc globular domains were replaced,respectively, with the corresponding sequences of human C1qa, C1qb, andC1qc. Alignments of human and rat C1q (and mouse) protein sequences aredepicted in FIGS. 3A, B, and C, where the boundaries for globular headsare as indicated, and the boundaries of rat/human genes are indicatedwith arrows. The amino acid sequences of the humanized rat C1qa, C1qb,and C1qc proteins are set forth in SEQ ID NOs: 55, 56, and 57,respectively, and are listed in Table 9, with rat sequences italicized.

TABLE 9 Amino Acid Sequences of the Chimeric Rat/Human C1q proteinsSEQ ID Protein Sequence NO: C1qaMETSQGWLVACVLAVTLVWTVAEDVCRAPNGKDGVAGIPG 55RPGRPGLKGERGEPGAAGIRTGIRGLKGDMGESGPPGKPGNVGFPGPTGPLGNSGPQGLKGVKGNPGNIRD QPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA* C1qbMKTQWSEILTPLLLLLLGLLHVSWAQSSCTGSPGIPGVPGIPG 56VPGSDGKPGTPGIKGEKGLPGLAGDHGELGEKGDAGIPGIPGKVGPKGPVGPKGAPGPPGPRGPKGDSGDYKAT QKIAFSATRTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA* C1qcMLRMVVGTSCQPQHGLYLLLLLLALPLRSQANAGCYGIPGMP 57GLPGTPGKDGHDGLQGPKGEPGIPAIPGTQGPKGQKGEPGMPGHRGKNGPMGTSGSPGDPGPRGPPGEPGEEGR YKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD*

Example 2.1 Generation of C1q Knock Out Rat

In detail, first, a rat C1q locus comprising all three C1q genes wasmodified to delete a 17.6 Kb nucleotide sequence comprising genesencoding rat C1q (see FIG. 2A). A targeting vector was synthesized tocomprise a LacZ gene and a self-deleting hygromycin selection cassette,and the vector contained 5′ and 3′ homology arms allowing deletion ofall three rat C1q genes from C1qa ATG to C1qb stop codon. The vectorcomprising the C1q sequence deletion was introduced into rat C1q BAC viabacterial homologous recombination (BHR), and the resultant BAC DNA wasused to electroporate rat ES cells to create modified ES cells forgenerating a rat that lacked the endogenous C1q locus. The sequences ofthe various junctions in the deleted locus are labeled in the secondschematic diagram in FIG. 2C, with corresponding nucleic acid sequenceslisted in Table 10 below. The junctions shown in Tables in this exampleonly list short nucleotide sequences, but direct one skilled in the artto the location where the sequences were inserted into the rat genome.

TABLE 10 Junction Sequences of the deletion of Rat C1q Locus JunctionSEQ Description Sequence ID NO Rat C1qaGATTCTCCCAATCTCTCCTCTGCAGGACCACTGGATCATT 58 promoter/LacTAAATCGGTACC/CATGATGTTCCTGCAGAGACACACA Z sequenceGGGACCCCGGGCATGCTGGACAGTCA LacZ TAGTTATCGAGCCCGGGGATCCACTAGTTCTAGTGTTTAA59 sequence/SD ACTCTAGCCG/GGGGATCCAGACATGATAAGATACATTGA C-loxP-HygTGAGTTTGGACAAACCACAACT cassette SDC-loxP-AGGATTACTGGCAGGGAGGAGGTTTTGGATAGGAGTGAT 60 HygTTGACCCCGTGA/GCTAGCATAACTTCGTATAGCATACATT cassette/3′ ofATACGAAGTTATCTAGGGGCTG rat C1q sequence

ES cells containing deletion of rat C1q sequences were identified by aquantitative TAQMAN™ assay modification of allele assay (MOA), describedabove. Table 11 identifies the names and locations of each ofprimers/probe sets used in the quantitative PCR assays. Same ES cellsare used to introduce human C1q sequences to generate a chimeric rat asdescribed below.

TABLE 11Primer and Probes Used in a MOA Assay to Confirm Deletion of Rat C1q locusLoss of Allele (LOA) or Gain of SEQ ID Description Sequence Allele (GOA)NO Rno C1qTU2 - AACCCACCGACGTATGGCAACGT LOA (rat C1qa) 61 Probe FGCCAGCTTTCTCAGCTATTCG 62 R GCGGTTCTGGTATGGATTCTC 63 Rno C1qTD-AAACACCTTCCAGGTCACCACGGG LOA (rat C1qb) 64 Probe F TCTCACCTTCTGCGACTATGC65 R CCTGCTCCAGCTTCAAGACTAC 66 Hyg-Probe ACGAGCGGGTTCGGCCCATTC GOA 67 FTGCGGCCGATCTTAGCC (hygromycin) 68 R TTGACCGATTCCTTGCGG 69 LacZ ProbeCGATACTGTCGTCGTCCCCTCAAACTG GOA (LacZ) 70 F GGAGTGCGATCTTCCTGAGG 71 RCGCATCGTAACCGTGCATC 72

Targeted chimeric C1q Dark Agouti ES cells are implanted into SpragueDawley rat embryos to generate F0 pups bearing deletion of C1q locus. F0chimeric pups are bred to wild type rats to create F1 pups that areheterozygous for the genetic manipulation; the presence of the modifiedallele is confirmed by TAQMAN® assay as described above. F1 pups aresubsequently bred to homozygosity.

Example 2.2. Generation of Humanized C1q Rat

To generate humanized C1q rat, plasmids were synthesized by Blue Heronusing sequences for human C1q globular head domains (the same sequencesas used for the humanized C1q mouse in Example 1 above) and overlappingrat sequences. For C1qb humanization construct, a self-deletingloxP-puromycin (SDC-loxp-Puro) selection cassette was inserted byrestriction digest downstream of the human C1qb polyA sequence. For bothC1qa and C1qc constructs, a spectinomycin (Spec) selection cassette wasinserted by restriction digest downstream of each C1qa and C1qc polyAsequences. The resultant constructs are introduced sequentially into theRat C1q BAC from LUCIGEN® through either a combination CRISPR/CAS9technology and Gibson Assembly or bacterial homologous recombination(BHR) followed by selection and/or digestion steps to remove theselection cassettes, as demonstrated in FIG. 2B. In this particularembodiment, the chimeric nucleic acid sequence for C1qb was introducedby BHR first (1 in the figure), followed by BHR of chimeric C1qc DNA (2in the figure), and then introduction by BHR of chimeric C1qa DNA (3 inthe figure).

The large targeting vector containing all three chimeric C1q genes waselectroporated into Rat C1q KO HET ES cells as depicted in FIG. 2C, andsuccessful integration was confirmed by a TAQMAN® real-time PCT-basedmodification of allele (MOA) assay. Primers and probes used for the MOAassay are described in Table 12.

TABLE 12Primer and Probes Used in MOA Assay to Confirm Presence of ChimericHuman/Rat C1q genes Loss of Allele (LOA) or Gain of SEQ ID DescriptionSequence Allele (GOA) NO Rno C1qTU2-Probe AACCCACCGACGTATGGCAACGTLOA (rat 73 F GCCAGCTTTCTCAGCTATTCG C1qa) 74 R GCGGTTCTGGTATGGATTCTC 75RnoC1qTD-Probe AAACACCTTCCAGGTCACCACGGG LOA (rat 76 FTCTCACCTTCTGCGACTATGC C1qb) 77 R CCTGCTCCAGCTTCAAGACTAC 781565ha2 -Probe CCTTCCAGGTGCTGTCCCAGTG GOA (human 79 FGTACCCGGCTACTACTACTTCA C1qa) 80 R GAGACGATGGACAGGCAGATTTC 81 1565hb2ACCATCAACGTCCCCCTGCGC GOA (human 82 F AATCGCCTTCTCTGCCACAA C1qb) 83 RGTGGTCGAAGCGGATGGT 84 1565hc4 CACCTGCAAAGTCCCCGGCCTC GOA (human 85 FTGACACGAGCACTGGCAAGT C1qc) 86 R CGACGCGTGGTAGACAAAGTAG 87

Junction sequences between various genetically engineered components atthe chimeric locus are depicted in Table 13 below, and are indicated onthe bottom schematic diagram in FIG. 2C. The junctions shown in Tablesin this example only list short nucleotide sequences, but direct oneskilled in the art to the location where the sequences were insertedinto the rat genome.

TABLE 13 Junction Sequences of the Chimeric Human/Rat C1q Locus JunctionSEQ ID Description Sequence NO Rat C1qa exonGCCCCCAAGGGTTGAAAGGTGTGAAAGGCAATCCGGGC 88 3/human C1QAAATATCAGGGA/CCAGCCGAGGCCAGCCTTCTCCGCCATT (globularCGGCGGAACCCCCCAATGGGGGGCA domain) Human C1QAGCATTGAGAGGGAGGCCTAAGAATAATAACAATCCA 89 3′UTR/3′ of ratGTGCTTAAGAGTCAGGC/GCTGGGTAGCTGCCCCACG C1qa (non-TTCTGCCATCTCCTGCACTCCCTGTTGCGGGGCC coding) Rat C1qc exonGGGATCCAGGCCCCAGGGGTCCTCCCGGGGAGCCG 90 3/human C1QCGGTGAGGAGGGTCG/ATACAAGCAGAAATTCCAGTC (globularAGTGTTCACGGTCACTCGGCAGACCCACCAGCCCC domain) Human C1QCTGAATTTCGGATCTTCAACTTTGCATCAGCCATAG 91 3′UTR/3′ of ratCTGGGCTCTGGACTC/GAATGGCAGGCTGGGTCCA C1qc (nonGCACCCGGACGCCCGCCTCGCTCCCTCTGCT coding) Rat C1qb exonGCCCCCCTGGACCCCGCGGTCCCAAAGGTGACTCTGG 92 3/human C1QBAGACTACAAGGCTACC/CAGAAAATCGCCTTCTCTGCCA (globularCAAGAACCATCAACGTCCCCCTGCGCCGG domain) Human C1QBCTGGCACACCAGAAGTGCCATGCTCAGAAATGTTGGTT 93 3′UTR/ SDC-ACATGAATGAAT/GTCGAGATAACTTCGTATAATGTATG loxP-puro regionCTATACGAAGTTATATGCATGCCAG SDC-loxP-puroGGCGGCCTAGATAACTTCGTATAATGTATGCTATACG 94 region/I-CeuAAGTTATGCTAGG/TAACTATAACGGTCCTAAG site/3′ of ratGTAGCGA/GCTAGCTCACGGGGTCAAATCACTCCTATC C1qb (non-CAAAACCTCCTCCCTGCCAGTAATCC coding)

Targeted chimeric C1q Dark Agouti ES cells are implanted into SpragueDawley rat embryos to generate F0 pups bearing the chimeric human/ratC1q locus. F0 chimeric pups are bred to wild type rats to create F1 pupsthat are heterozygous for the genetic manipulation; the presence of themodified allele is confirmed by TAQMAN® assay as described above. F1pups are subsequently bred to homozygosity.

Example 3: Characterization of Humanized C1q Mouse Example 3.1: ChimericC1q Is Present and Functional in Mouse Serum

In order to determine if chimeric C1q was expressed and functional inmouse serum, humanized C1q mice were phenotyped by Western blot andclassical complement hemolysis assay. All mice were housed and bred inthe specific pathogen-free facility at Regeneron Pharmaceuticals. Allanimal experiments were approved by IACUC and Regeneron Pharmaceuticals.

(1) Western Blot:

Serum C1q concentrations were assayed in 1615 HO mice (mice homozygousfor humanized C1q as described above) using Western blot, as follows:mouse or normal human serum (NHS) were diluted in PBS. Normal humanserum (Quidel) was used as a positive control. Serum was added toelectorophoresis sample loading buffer containing mercaptoethanol andSDS and run on a polyacrylamide gel under reducing/denaturingconditions, then transferred onto nitrocellulose membrane. Blots wereblocked, then probed with goat anti-human C1q primary antibody (Quidel),followed by detection with donkey anti-goat IgG HRP (Santa Cruz).ThermoScientific Super Signal West Pico Chemiluminescent Subtrate wasused to develop the blot. GE Image Quant LAS4000 was used for imaging.

(2) Classical Pathway Hemolysis Assay:

Desired number of SRBC (sheep red blood cells) were washed in GVB++buffer and re suspended at 1×10⁹ cells/mL and opsonized with rabbitanti-sheep hemolysin. Sensitized SRBC were diluted to 2×10⁸ cells/mL inGVB++ buffer prior to using in hemolysis assay. Serum from WTlittermates (n=5) and 1615HO (n=4) mice was collected at seven to nineweeks of age. Mouse serum was serially diluted in a 6 point, 2-folddilution series from 1/5 to 1/160 with GVB++ buffer (100 ul dilutedserum/well). Immediately, 100 uL of sensitized SRBCs (at 2×10⁸ cells/mL)were added, for a total volume of 200 uL, and incubated 1 hr at 37° C.After the incubation time, cells were spun down by centrifugation at1250×g at 4° C. A total of 100 uL of the supernatant was transferred toa fresh 96-well flat bottom plate and read at 541 nm on a MolecularDevices Spectramax M5 microplate reader and SoftMax Pro software. Thehemolytic activity was calculated: OD541 of all experimental samples wasdivided by the OD541 at Maximum cell lysis (cells treated with 100 uLwater) and then multiplied by 100. Data represented are single points(duplicates not run).

As demonstrated in FIG. 4, top panel, chimeric C1q proteins, as detectedby anti-human C1q antibody, were detected in the serum of humanized C1qmice, albeit less C1q protein was detected in humanized C1q mouse serumthan in human serum. The chimeric C1q protein obtained from thehumanized mouse displayed similar classical complement activity asmeasured by hemolysis assay to that observed in the mice comprising wildtype mouse C1q (FIG. 4, bottom panel).

The concentration of chimeric human/mouse C1q in mouse serum was alsodetermined using a sandwich ELISA format with antibodies specific forhuman head of C1q. The standard curve was generated using a knownconcentration of human C1q protein, and the concentration of chimericC1q in mouse serum was determined to be in the range of about 10-30ug/mL.

Example 4: Humanized C1q Mouse as a Model for Testing Human Therapeutics

To confirm whether the humanized C1q mouse can serve as a model fortesting human therapeutics, we first developed an in vitro assay toassess the ability of humanized C1q to activate complement dependentcytotoxicity (CDC) of Raji cells (B cells expressing cell-surfaceantigen CD20) by binding to human anti-CD20 antibody. Therapeuticanti-CD20 antibodies against the B-cell specific cell-surface antigenCD20 have been shown to lead to CDC of B-cells (Glennie et al. 2007,Mechanisms of killing by anti-CD20 monoclonal antibodies, Mol. Immunol.,Vol. 44(16) pp. 3823-3′7) and CDC assay using cell lines expressing CD20has been described previously (Flieger et al. 2000, Mechanisms ofCytotoxicity Induced by Chimeric Mouse Human Monoclonal AntibodyIDEC-C2BB in CD20-Expressing Lymphoma Lines, Cell Immunol., Vol. 204(1)pp. 55-63).

For the CDC bioassay, Raji cells were seeded onto a 96-well assay platesat 10,000 cells/well in 1% BSA containing RPMI 1640. To measure CDC withhuman or mouse serum (from either humanized C1q or WT mice), the humananti-CD20 antibody was diluted 1:4 from 2 nM to 0.007 nM and incubatedwith cells for 10 minutes at 25° C. At the conclusion of the incubationwith the anti-CD20 antibody, the serum was added to cells at a finalconcentration of 1%. Cytotoxicity was measured after 1 hour ofincubation at 37° C. and in 5% CO2, followed by a 30 minute incubationat 25° C., and addition of CytoTox-Glo™ reagent (Promega, #G9291).CytoTox-Glo™ is a luminescence-based reagent that measures cell killingsuch that increased luminescence is observed with increased cytotoxicity(measured in relative light units, RLUs). Untreated cells in controlwells were lysed by treatment with digitonin 10 minutes before additionof CytoTox-Glo™ reagent to determine maximal killing of cells. Plateswere read for luminescence by a Victor X instrument (Perkin Elmer) 10-15minutes following the addition of CytoTox-Glo™. Where calculated, thepercentage of cytotoxicity was calculated with the RLU values by usingthe following equation:

${\%\mspace{14mu}{Cytoxicity}} = {100 \times \frac{\left( {{{Experimental}\mspace{14mu}{Cell}\mspace{14mu}{Lysis}} - {{Background}\mspace{14mu}{Cell}\mspace{14mu}{Lysis}}} \right)}{\left( {{{MaximumCell}\mspace{14mu}{Lysis}} - {{Background}\mspace{14mu}{Cell}\mspace{14mu}{Lysis}}} \right)}}$

In this equation “background cell lysis” is the luminescence from thecells treated with media and serum alone without any anti-CD20 antibodyand the “maximum cell lysis” is the luminescence from the cells treatedwith digitonin. The results, expressed as % cytotoxicity or RLUs, wereanalyzed using nonlinear regression (4-parameter logistics) with Prism 7software (GraphPad).

Complement dependent cytotoxicity (CDC) activity mediated by 2 nM humananti-CD20 antibody and normal human serum resulted in 83% of maximumlysis, while CDC mediated by 2 nM human CD20 antibody and serum fromhumanized C1q mice was 55-58% of maximum lysis (FIG. 5). No cell lysiswas detected using wild type mouse serum. Thus, serum containinghumanized C1q globular head led to more efficient complement dependentlysis of Raji cells mediated by the human anti-CD20 antibody compared tothe serum containing wild-type mouse C1q protein.

For in vivo testing, S. aureus infection model was chosen. S. aureus isa major cause of bacteremia in patients, and these infections are oftenfatal. Mouse models of bacteremia have been developed by manylaboratories using a variety of laboratory-adapted and clinical S.aureus isolates and in a variety of mouse backgrounds (O'Keeffe K M, etal, Infect Immun. 2015 September; 83(9):3445-57. Manipulation ofAutophagy in Phagocytes Facilitates Staphylococcus aureus BloodstreamInfection; Rauch et al, Infect Immun. 2012 October; 80(10):3721-32.Abscess formation and alpha-hemolysin induced toxicity in a mouse modelof Staphylococcus aureus peritoneal infection) to study the infectiondynamics and effect of potential therapeutics. A bacteremia model wasestablished to evaluate the activity of bispecific antibodies (bisAbs),with one arm targeting C1q and another arm targeting an antigenexpressed on S. aureus, in both reducing bacterial burden and improvingsurvival.

Humanized C1q mice and control wild type mice were infectedintraperitoneally with 1.7×10⁸ colony forming units (CFUs) per mouse ina 200 ul volume of S. aureus Newman grown to log phase, OD₆₀₀≤1, in TSBat 37° C. and washed 3 times in PBS. Immediately following infection,mice were dosed with 100 ug of each bisAb and isotype-matched antibodyin a 100 ul volume, intraperitoneally. Mice were euthanized on Day 3 andkidneys were collected to determine organ burden. Briefly, kidneys werehomogenized in 5.0 mL PBS using gentleMACS Octo Dissociator (MiltenyiBiotec). Tissue homogenate was diluted in PBS and multiples of 10-foldserial dilutions were plated on LB agar plates and incubated at 37° C.overnight. Next day individual colonies were counted to determinebacterial burden at that time point post-infection and results werereported as CFUs/gram tissue.

BisAbs were tested alongside an isotype control antibody for efficacy inreducing bacterial kidney burden in female humanized C1q mice and WTcontrol mice. Compared to mice treated with isotype control Ab, on day 3after treatment, 2-4 log reduction of bacterial CFUs was detected inkidneys of bisAbs treated humanized C1q mice; however, similar toresults obtained with isotype control antibody, no reduction inbacterial burden was observed in WT mice expressing endogenous mouse C1qtreated with BisAbs (data not shown).

To examine the effect of the bisAbs on survival, humanized C1q mice wereinfected with 1.5×10⁸ CFUs per mouse of S. aureus Newman and treatedwith test antibodies as described above. Instead of sacrificing the miceon Day 3, they were monitored until Day 18. Mice that lost 20% of theirstarting body weight were sacrificed and recorded as a death. Thepercentage of surviving animals was reported at the end of the study.

BisAb was tested alongside an isotype control antibody for efficacy in asurvival study in humanized C1q female mice (n=9). As shown in Table 14,at the end of the study, on Day 18 post infection, 100% of the micetreated with BisAb survived, in comparison to 78% of the isotype controltreated mice.

TABLE 14 Survival of Humanized C1q Mice in Bacteremia Model Survival atD18 post- Ab infection (%) Mice (n) BisAb 100 9 Isotype Control 78 9Sham (PBS) 56 9

In conclusion, this study demonstrates that humanized C1q mice are avaluable model for testing therapeutic agents, such as antibodies (e.g.,bispecific antibodies), which are directed against human C1q protein.

Example 5: Characterization of Humanized C1q Rat

In order to determine if chimeric C1q was functional in rat serum,humanized C1q rats described in Example 2 were phenotyped by classicalcomplement hemolysis assay. Results were compared to C1q knock-out (KO)rats and normal human serum. All rats were 50% Dark Agouti 50% SpragueDawley background. All rats were housed and bred in the specificpathogen-free facility at Regeneron Pharmaceuticals. All animalexperiments were approved by IACUC and Regeneron Pharmaceuticals.

(1) Classical Pathway Hemolysis Assay

Desired number of SRBCs (sheep red blood cells) were washed in GVB++buffer and re suspended at 1×10⁹ cells/mL and opsonized with rabbitanti-sheep hemolysin. Sensitized SRBCs were diluted to 2×10⁸ cells/mL inGVB++ buffer prior to using in hemolysis assay. Serum from WT (n=3females and n=4 males), 100015 HO (homozygous humanized C1q rats) (n=5females and n=6 males) and homozygous C1q knock-out rats (n=2 females)was collected at ten to seventeen weeks of age. Normal human serum(Quidel) was used as a positive control, while C1q depleted human serum(Quidel) was used as a negative control. Rat and human serum wasserially diluted in a 12 point, 2-fold dilution series from 1/5 to1/10240 with GVB++ buffer (100 ul diluted serum/well). Immediately, 100uL of sensitized SRBCs (at 2×10⁸ cells/mL) were added, for a totalvolume of 200 uL, and incubated 1 hr at 37° C. After the incubationtime, cells were spun down by centrifugation at 1250×g at 4° C. A totalof 100 uL of the supernatant was transferred to a fresh 96-well flatbottom plate and read at 412 nm on a Molecular Devices Spectramax M5microplate reader and SoftMax Pro software. The hemolytic activity wascalculated: OD541 of all experimental samples was divided by the OD541at Maximum cell lysis (cells treated with 100 uL water) and thenmultiplied by 100. Data represented are single points (duplicates notrun).

As shown in FIG. 6, the chimeric C1q protein obtained from humanizedrats displayed similar classical complement activity as measured byhemolysis assay to that observed in the rats comprising wild type ratC1q, and to that observed with normal human serum. No differences wereobserved between male and female rats.

Example 6: Humanized C1q Rat as a Model for Testing Human Therapeutics

To confirm whether a humanized C1q rat can serve as a model for testinghuman therapeutics, an in vitro complement dependent cytotoxicity assayand a whole blood bacterial survival assay were performed.

(1) Complement Dependent Cytotoxicity (CDC) Assay

For the CDC bioassay, Raji cells (a human B cell line expressing CD20),a serum sample (complement preserved human serum, WT rat serum, or serumfrom a homozygous C1q humanized rat as described in Example 2), and ananti-CD20 antibody, were used.

Raji cells were seeded onto a 96-well assay plates at 10,000 cells/wellin 1% BSA containing RPMI 1640. To measure CDC with human or rat serum,the anti-CD20 antibody was diluted 1:4 from 20 nM to 0.019 nM (includinga control sample containing no antibody) and incubated with cells for 10minutes at 25° C. followed by addition of 0.5% serum. Cytotoxicity wasmeasured after 1 hour of incubation at 37° C. and in 5% CO2, followed by30 minute incubation at 25° C., and addition of CytoTox-Glo™ reagent(Promega, #G9291). CytoTox-Glo™ is a luminescence-based reagent thatmeasures cell killing such that increased luminescence is observed withincreased cytotoxicity (measured in relative light units, RLUs).Untreated cells in control wells were lysed by treatment with digitoninimmediately after addition of CytoTox-Glo™ reagent to determine maximalkilling of cells. Plates were read for luminescence by a Victor Xinstrument (Perkin Elmer) 10-15 minutes following the addition ofCytoTox-Glo™. Where calculated, the percentage of cytotoxicity wascalculated with the RLU values by using the following equation:

${\%\mspace{14mu}{Cytoxicity}} = {100 \times \frac{\left( {{{Experimental}\mspace{14mu}{Cell}\mspace{14mu}{Lysis}} - {{Background}\mspace{14mu}{Cell}\mspace{14mu}{Lysis}}} \right)}{\left( {{{MaximumCell}\mspace{14mu}{Lysis}} - {{Background}\mspace{14mu}{Cell}\mspace{14mu}{Lysis}}} \right)}}$

In this equation “background cell lysis” is the luminescence from thecells treated with media and serum alone without any anti-CD20 antibodyand the “maximum cell lysis” is the luminescence from the cells treatedwith digitonin. The results, expressed as cytotoxicity or RLUs, wereanalyzed using nonlinear regression (4-parameter logistics) with Prism 7software (GraphPad).

As shown in FIG. 7, complement dependent cytotoxicity activity mediatedby 20 nM CD20 antibody and the humanized C1q rat (MAID10015) serumresulted in 85-90% maximum lysis of Raji cells, which was similar tolysis using wild type rat serum (93-112% lysis) and normal human serum(83% lysis). Humanization of the C1q molecule did not alter thecomplement dependent lysis of Raji cells mediated by the CD20 antibodyas compared to the rat C1q.

(2) S. aureus Survival in C1q Humanized Rat Blood and the Effects of aBispecific Antibody

S. aureus survival in whole human blood can be assessed in an ex vivoassay to explore the role of complement and immune effector cells inmodulating bacterial growth (Thammavongsa et al., J Exp Med. 2009Oct.26; 206(11):2417-27). In this assay, the activity of a bispecificantibody targeting C1q and a S. aureus antigen in modulating S. aureussurvival is measured where the bispecific antibody is added into wholeblood and survival is assessed after 24 hours. This assay has beenadapted in this Example to use C1q humanized rat blood and examine thefunctionality of the humanized chimeric C1q protein in mediating theeffects of a bispecific antibody that specifically recognizes a S.aureus antigen and the human or humanized C1q protein but not the ratC1q protein. A bivalent monospecific antibody against the same S. aureusantigen was used as a control.

Briefly, a culture of S. aureus Newman was grown in RPMI overnight,washed in PBS, and resuspended to a concentration of 1.25×10⁸ colonyforming units (CFU)/mL in PBS and serially diluted to a concentration of10⁵ CFU/mL. In duplicates, 10⁴ CFU of the S. aureus suspension was mixedwith 100 ug/mL bispecific antibody, control antibody or no antibody and100 uL of humanized C1q or wild type (WT) rat blood (in sodium citrateas anti-coagulant with additional 500 nM dabigatran to prevent clotformation). The samples were incubated in 96 well plates at 37° C. withshaking (100 rpm) for 24 hours. After incubation, 100 ul ofagglutination lysis buffer (PBS supplemented with 200U Streptokinase, 2ug/mL RNase, 10ug/mL DNase, 0.5% saponin, 100 ug trypsin per ml of PBS)was added to the samples and vigorously vortexed until the pelletdisappeared. A total of 50 uL from each sample was serially diluted inPBS and plated onto LB agar plates for enumeration of CFUs.

In this assay, freshly drawn blood samples from 11 humanized C1q and 10WT rats were tested. Percent survival of S. aureus after treatment withthe bispecific or control antibodies was determined. The overall growthin rat blood in the absence of test antibody is normalized to 100%.Survival of S. aureus in the WT rat blood with the bispecific antibodytreatment ranged from 58-131%, while the bispecific antibody treatmentin C1q humanized rat blood resulted in 7-49% survival. Survival of S.aureus in the WT rat blood with the control antibody treatment rangedfrom 40-139%, and the control antibody treatment in C1q humanized ratblood resulted in 61-114% survival.

What is claimed is:
 1. A genetically modified non-human animal comprising in its genome a nucleic acid encoding a chimeric C1q polypeptide, wherein the nucleic acid comprises a non-human nucleic acid sequence and a human nucleic acid sequence, wherein the chimeric C1q polypeptide is selected from the group consisting of a chimeric C1qa polypeptide, a chimeric C1qb polypeptide, and a chimeric C1qc polypeptide, and wherein the chimeric C1q polypeptide comprises a globular head domain that is substantially human and an N-terminal stalk-stem region that is substantially non-human.
 2. The genetically modified non-human animal of claim 1, wherein the non-human animal expresses a chimeric C1qa polypeptide, a chimeric C1qb polypeptide, a chimeric C1qc polypeptide, or a combination thereof.
 3. The genetically modified non-human animal of claim 1 or 2, wherein the non-human animal is a mammal.
 4. The genetically modified non-human animal of claim 1 or 2, wherein the non-human animal is a rodent.
 5. The genetically modified non-human animal of claim 1 or 2, wherein the non-human animal is a rat or a mouse.
 6. The genetically modified non-human animal of any one of claims 1-5, wherein the chimeric C1q polypeptide is a chimeric C1qa polypeptide.
 7. The genetically modified non-human animal of claim 6, wherein the chimeric C1qa polypeptide comprises a globular head domain that is substantially identical to the globular head domain of a human C1qa polypeptide, and an N-terminal stalk-stem region that is substantially identical to the N-terminal stalk-stem region of a non-human C1qa polypeptide.
 8. The genetically modified non-human animal of claim 7, wherein the globular head domain of a human C1qa polypeptide comprises 108-245 of SEQ ID NO:
 4. 9. The genetically modified non-human animal of any one of claims 6-8, wherein the non-human animal is a mouse.
 10. The genetically modified non-human animal of claim 9, wherein the chimeric C1qa polypeptide comprises an N-terminal stalk-stem region substantially identical to the N-terminal stalk-stem region of the endogenous mouse C1qa polypeptide, and wherein the N-terminal stalk-stem region of the endogenous mouse C1qa polypeptide comprises amino acids 23-107 of SEQ ID NO:
 1. 11. The genetically modified non-human animal of claim 9 or 10, wherein the chimeric C1qa polypeptide comprises amino acids 23-245 of SEQ ID NO:
 10. 12. The genetically modified non-human animal of any one of claims 6-8, wherein the non-human animal is a rat.
 13. The genetically modified non-human animal of claim 12, wherein the chimeric C1qa polypeptide comprises an N-terminal stalk-stem region substantially identical to the N-terminal stalk-stem region of the endogenous rat C1qa polypeptide, and wherein the N-terminal stalk-stem region of the endogenous rat C1qa polypeptide comprises amino acids 23-107 of SEQ ID NO:
 7. 14. The genetically modified non-human animal of claim 12 or 13, wherein the chimeric C1qa polypeptide comprises amino acids 23-245 of SEQ ID NO:
 55. 15. The genetically modified non-human animal of any one of claims 1-5, wherein the chimeric C1q polypeptide is a chimeric C1qb polypeptide.
 16. The genetically modified non-human animal of claim 15, wherein the chimeric C1qb polypeptide comprises a globular head domain that is substantially identical to the globular head domain of a human C1qb polypeptide, and an N-terminal stalk-stem region that is substantially identitical to the N-terminal stalk-stem region of a non-human C1qb polypeptide.
 17. The genetically modified non-human animal of claim 16, wherein the globular head domain of a human C1qb polypeptide comprises amino acids 115-251 of SEQ ID NO:
 5. 18. The genetically modified non-human animal of any one of claims 15-17, wherein the non-human animal is a mouse.
 19. The genetically modified non-human animal of claim 18, wherein the chimeric C1qb polypeptide comprises an N-terminal stalk-stem region substantially identical to the N-terminal stalk-stem region of the endogenous mouse C1qb polypeptide, and wherein the N-terminal stalk-stem region of the endogenous mouse C1qb polypeptide comprises amino acids 26-114 of SEQ ID NO:
 2. 20. The genetically modified non-human animal of claim 18 or 19, wherein the chimeric C1qb polypeptide comprises amino acids 26-251 of SEQ ID NO:
 11. 21. The genetically modified non-human animal of any one of claims 15-17, wherein the non-human animal is a rat.
 22. The genetically modified non-human animal of claim 21, wherein the chimeric C1qb polypeptide comprises an N-terminal stalk-stem region substantially identical to the N-terminal stalk-stem region of the endogenous rat C1qb polypeptide, and wherein the N-terminal stalk-stem region of the endogenous rat C1qb polypeptide comprises amino acids 26-114 of SEQ ID NO:
 8. 23. The genetically modified non-human animal of claim 21 or 22, wherein the chimeric C1qb polypeptide comprises amino acids 26-251 of SEQ ID NO:
 56. 24. The genetically modified non-human animal of any one of claims 1-5, wherein the chimeric C1q polypeptide is a chimeric C1qc polypeptide.
 25. The genetically modified non-human animal of claim 24, wherein the chimeric C1qc polypeptide comprises a globular head domain that is substantially identical to the globular head domain of a human C1qc polypeptide, and an N-terminal stalk-stem region that is substantially identical to the N-terminal stalk-stem region of a non-human C1qc polypeptide.
 26. The genetically modified non-human animal of claim 25, wherein the globular head domain of a human C1qc polypeptide comprises amino acids 113-245 of SEQ ID NO:
 6. 27. The genetically modified non-human animal of any one of claims 24-26, wherein the non-human animal is a mouse.
 28. The genetically modified non-human animal of claim 27, wherein the chimeric C1qc polypeptide comprises an N-terminal stalk-stem region substantially identical to the N-terminal stalk-stem region of the endogenous mouse C1qc polypeptide, and wherein the N-terminal stalk-stem region of the endogenous mouse C1qc polypeptide comprises amino acids 30-113 of SEQ ID NO:
 3. 29. The genetically modified non-human animal of claim 27 or 28, wherein the chimeric C1qc polypeptide comprises amino acids 30-246 of SEQ ID NO:
 11. 30. The genetically modified non-human animal of any one of claims 24-26, wherein the non-human animal is a rat.
 31. The genetically modified non-human animal of claim 30, wherein the chimeric C1qc polypeptide comprises an N-terminal stalk-stem region substantially identical to the N-terminal stalk-stem region of the endogenous rat C1qc polypeptide, and wherein the N-terminal stalk-stem region of the endogenous rat C1qc polypeptide comprises amino acids 32-115 of SEQ ID NO:
 9. 32. The genetically modified non-human animal of claim 30 or 31, wherein the chimeric C1qc polypeptide comprises amino acids 32-248 SEQ ID NO:
 57. 33. The genetically modified non-human animal of any of the preceding claims, wherein the chimeric C1q polypeptide comprises a non-human C1q signal peptide, optionally an endogenous non-human C1q signal peptide.
 34. The genetically modified non-human animal of claim 1, wherein the nucleic acid encoding the chimeric C1q polypeptide is at an endogenous non-human C1q locus.
 35. The genetically modified non-human animal of claim 34, wherein an endogenous genomic sequence at the endogenous non-human C1q locus has been replaced by the human nucleic acid sequence.
 36. The genetically modified non-human animal of any one of claim 1, 2, 34 or 35, wherein the human nucleic acid sequence encodes substantially the globular head domain of a human C1q polypeptide.
 37. The genetically modified non-human animal of claim 36, wherein the human nucleic acid sequence is a genomic fragment of a human C1q gene.
 38. The genetically modified non-human animal of claim 37, wherein the genomic fragment comprises the 3′ UTR of the human C1q gene.
 39. The genetically modified non-human animal of any of claims 36-38, wherein the chimeric C1q polypeptide is a chimeric C1qa polypeptide, and the human nucleic acid sequence encodes substantially the globular head domain of a human C1qa polypeptide.
 40. The genetically modified non-human animal of claim 39, wherein the globular head domain of the human C1qa polypeptide comprises amino acids 108-245 of SEQ ID NO:
 4. 41. The genetically modified non-human animal of claim 39 or 40, wherein the human nucleic acid sequence encodes amino acids 112-245 of SEQ ID NO:
 4. 42. The genetically modified non-human animal of any of claims 36-38, wherein the chimeric C1q polypeptide is a chimeric C1qb polypeptide, and the human nucleic acid sequence encodes substantially the globular head domain of a human C1qb polypeptide.
 43. The genetically modified non-human animal of claim 42, wherein the globular head domain of the human C1qb polypeptide comprises amino acids 115-251 of SEQ ID NO:
 5. 44. The genetically modified non-human animal of claim 42 or 43, wherein the human nucleic acid sequence encodes amino acids 118-251 of SEQ ID NO:
 5. 45. The genetically modified non-human animal of any of claims 36-38, wherein the chimeric C1q polypeptide is a chimeric C1qc polypeptide, and the human nucleic acid sequence encodes substantially the globular head domain of a human C1qc polypeptide.
 46. The genetically modified non-human animal of claim 45, wherein the globular head domain of the human C1qc polypeptide comprises amino acids 113-245 of SEQ ID NO:
 6. 47. The genetically modified non-human animal of claim 45 or 46, wherein the human nucleic acid sequence encodes amino acids 114-245 of SEQ ID NO:
 6. 48. The genetically modified non-human animal of any one of claim 1, 2, or 34-36, wherein the non-human nucleic acid sequence encodes substantially the N-terminal stalk-stem region of a non-human C1q polypeptide.
 49. The genetically modified non-human animal of claim 48, wherein the the non-human animal is a mouse, and the N-terminal stalk-stem region of mouse C1q polypeptide comprises amino acids 23-107 of SEQ ID NO: 1 (for C1qa), amino acids 26-114 of SEQ ID NO: 2 (for C1qb), or amino acids 30-113 of SEQ ID NO: 3 (for C1qc), respectively.
 50. The genetically modified non-human animal of claim 49, wherein the mouse comprises a nucleic acid encoding a chimeric C1qa polypeptide, wherein the nucleic acid comprises a mouse nucleic acid sequence and a human nucleic acid sequence, wherein the mouse nucleic acid sequence encodes amino acids 23-111 of SEQ ID NO:
 1. 51. The genetically modified non-human animal of claim 49, wherein the mouse comprises a nucleic acid encoding a chimeric C1qb polypeptide, wherein the nucleic acid comprises a mouse nucleic acid sequence and a human nucleic acid sequence, wherein the mouse nucleic acid sequence encodes amino acids 26-117 of SEQ ID NO:
 2. 52. The genetically modified non-human animal of claim 49, wherein the mouse comprises a nucleic acid encoding a chimeric C1qc polypeptide, wherein the nucleic acid comprises a mouse nucleic acid sequence and a human nucleic acid sequence, wherein the mouse nucleic acid sequence encodes amino acids 30-114 of SEQ ID NO:
 3. 53. The genetically modified non-human animal of claim 48, wherein the the non-human animal is a rat, and the N-terminal stalk-stem region of the endogenous C1q polypeptides comprises amino acids 23-107 of SEQ ID NO: 7 (for C1qa), amino acids 26-114 of SEQ ID NO: 8 (for C1qb), and amino acids 32-115 of SEQ ID NO: 9 (for C1qc), respectively.
 54. The genetically modified non-human animal of claim 53, wherein the rat comprises a nucleic acid encoding a chimeric C1qa polypeptide, wherein the nucleic acid comprises a rat nucleic acid sequence and a human nucleic acid sequence, wherein the rat nucleic acid sequence encodes amino acids 23-111 of SEQ ID NO:
 7. 55. The genetically modified non-human animal of claim 53, wherein the rat comprises a nucleic acid encoding a chimeric C1qb polypeptide, wherein the nucleic acid comprises a rat nucleic acid sequence and a human nucleic acid sequence, wherein the rat nucleic acid sequence encodes amino acids 26-117 of SEQ ID NO:
 8. 56. The genetically modified non-human animal of claim 53, wherein the rat comprises a nucleic acid encoding a chimeric C1qc polypeptide, wherein the nucleic acid comprises a rat nucleic acid sequence and a human nucleic acid sequence, wherein the rat nucleic acid sequence encodes amino acids 32-116 of SEQ ID NO:
 9. 57. The genetically modified non-human animal of claim 1, wherein the animal is a rat and comprises in its genome: a. at the endogenous C1qa locus a nucleic acid sequence encoding a chimeric rat/human C1qa polypeptide wherein the nucleic acid sequence comprises, 5′-3′ and in operable linkage a first nucleotide sequence encoding amino acids 1-111 of a rat C1qa polypeptide of SEQ ID NO: 7 and a second nucleotide sequence encoding amino acids 112-245 of a human C1qa polypeptide of SEQ ID NO: 4; b. at the endogenous C1qb locus a nucleic acid sequence encoding a chimeric rat/human C1qb polypeptide wherein the nucleic acid sequence comprises, 5′-3′ and in operable linkage a third nucleotide sequence encoding amino acids 1-117 of a rat C1qb polypeptide of SEQ ID NO: 8 and a fourth nucleotide sequence encoding amino acids 118-251 of a human C1qb polypeptide of SEQ ID NO: 5; and c. at the endogenous C1qc locus a nucleic acid sequence encoding a chimeric rat/human C1qc polypeptide wherein the nucleic acid sequence comprises, 5′-3′ and in operable linkage a fifth nucleotide sequence encoding amino acids 1-116 of a rat C1qc polypeptide of SEQ ID NO: 9 and a sixth nucleotide sequence encoding amino acids 114-245 of a human C1qc polypeptide of SEQ ID NO:
 6. 58. The genetically modified non-human animal of claim 1, wherein the animal is a mouse and comprises in its genome: a. at the endogenous C1qa locus a nucleic acid sequence encoding a chimeric mouse/human C1qa polypeptide wherein the nucleic acid sequence comprises, 5′-3′ and in operable linkage a first nucleotide sequence encoding amino acids 1-111 of a mouse C1qa polypeptide of SEQ ID NO: 1 and a second nucleotide sequence encoding amino acids 112-245 of a human C1qa polypeptide of SEQ ID NO: 4; b. at the endogenous C1qb locus a nucleic acid sequence encoding a chimeric mouse/human C1qb polypeptide wherein the nucleic acid sequence comprises, 5′-3′ and in operable linkage a third nucleotide sequence encoding amino acids 1-117 of a mouse C1qb polypeptide of SEQ ID NO: 2 and a fourth nucleotide sequence encoding amino acids 118-251 of a human C1qb polypeptide of SEQ ID NO: 5; and c. at the endogenous C1qc locus a nucleic acid sequence encoding a chimeric mouse/human C1qc polypeptide wherein the nucleic acid sequence comprises, 5′-3′ and in operable linkage a fifth nucleotide sequence encoding amino acids 1-114 of a mouse C1qc polypeptide of SEQ ID NO: 3 and a sixth nucleotide sequence encoding amino acids 114-245 of a human C1qc polypeptide of SEQ ID NO:
 6. 59. The genetically modified non-human animal of any one of claims 1-58, wherein the non-human animal does not express a functional endogenous C1qa, C1qb, and/or C1qc polypeptide(s).
 60. A method of making a genetically modified non-human animal, comprising modifying the genome of a non-human animal to comprise a nucleic acid encoding a chimeric C1q polypeptide, wherein the nucleic acid comprises a non-human nucleic acid sequence and a human nucleic acid sequence, wherein the chimeric C1q polypeptide is selected from the group consisting of a chimeric C1qa polypeptide, a chimeric C1qb polypeptide, a chimeric C1qc polypeptide, and a combination thereof, and wherein the chimeric C1q polypeptide comprises a globular head domain that is substantially human and an N-terminal stalk-stem region that is substantially non-human.
 61. The method of claim 60, wherein the genome of the non-human animal is modified to comprise a nucleic acid encoding a chimeric C1qa polypeptide, a nucleic acid encoding a chimeric C1qb polypeptide, and a nucleic acid encoding a chimeric C1qc polypeptide.
 62. The method of claim 60 or 61, wherein the nucleic acid encoding a chimeric C1q polypeptide is introduced at the endogenous non-human C1q locus.
 63. The method of claim 62, wherein the nucleic acid encoding a chimeric C1q polypeptide replaces a nucleotide sequence of the endogenous non-human C1q gene at the endogenous non-human C1q locus.
 64. The method of any one of claims 60-63, wherein the animal is a rodent, such as a mouse or a rat.
 65. The method of claim 64, wherein said modifying comprises a. introducing a nucleic acid molecule comprising the human nucleic acid sequence into the genome of a rodent embryonic stem (ES) cell, b. obtaining a rodent ES cell in which the human nucleic acid sequence is integrated into an endogenous C1q locus in an operable linkage to the endogenous non-human nucleic acid sequence so as to encode said chimeric C1q polypeptide, and c. generating a rodent animal from the rodent ES cell obtained in b.
 66. The method of claim 65, wherein the nucleic acid molecule comprises a nucleic acid encoding a chimeric C1qa polypeptide, a nucleic acid encoding a chimeric C1qb polypeptide, and a nucleic acid encoding a chimeric C1qc polypeptide.
 67. A chimeric C1q polypeptide comprising a globular head domain that is substantially human and an N-terminal stalk-stem region that is substantially non-human, wherein the chimeric C1q polypeptide is selected from the group consisting of a chimeric C1qa polypeptide, a chimeric C1qb polypeptide, and a chimeric C1qc polypeptide.
 68. A chimeric C1q polypeptide made from the non-human animal of any of claims 1-59.
 69. A chimeric C1q protein comprising one or more of the chimeric C1q polypeptides of claim 67 or
 68. 70. The chimeric C1q protein of claim 69, wherein the protein comprises at least one chimeric C1qa, at least one chimeric C1qb, and at least one chimeric C1qc polypeptide.
 71. The chimeric C1q protein of claim 70, wherein the protein comprises 6 each of the chimeric C1qa polypeptide, chimeric C1qb polypeptide, and chimeric C1qc polypeptide.
 72. An isolated nucleic acid encoding a functional chimeric C1q polypeptide comprising a non-human mammal nucleic acid sequence and a human nucleic acid sequence, wherein the human nucleic acid sequence encodes substantially the globular head domain of a human C1q polypeptide and the non-human nucleic acid sequence encodes substantially the N-terminal stalk-stem region of a cognate non-human C1q polypeptide, and wherein the C1q polypeptide is selected from a C1qa polypeptide, a C1qb polypeptide or a C1qc polypeptide.
 73. An isolated nucleic acid encoding a chimeric non-human mammal C1q protein, comprising one or more of a first, second or third nucleotide sequences, wherein: a. the first nucleotide sequence encodes a chimeric C1qa polypeptide, b. the second nucleotide sequence encodes a chimeric C1qb polypeptide, and c. the third nucleotide sequence encodes a chimeric C1qc polypeptide, wherein each of the first, second, and third nucleotide sequences comprises a non-human mammal nucleic acid sequence and a human nucleic acid sequence, wherein the human nucleic acid sequence encodes substantially the globular head domain of a human C1qa, C1qb, and C1qc polypeptide, respectively, and the non-human nucleic acid sequence encodes substantially the N-terminal stalk-stem region of a cognate non-human C1q polypeptide C1qa, C1qb, and C1qc polypeptide, respectively.
 74. A cell comprising an isolated nucleic acid of claim 73 or
 74. 75. The cell of claim 74, wherein the cell is an embryonic stem cell.
 76. A genetically modified non-human animal comprising the cell of claim
 75. 77. A transgenic rodent model for testing a C1q-based bi-specific antigen-binding protein, wherein the antigen-binding protein binds both human C1q and a non-rodent antigen of interest, comprising a genetically modified rodent of any of claims 1-59, and further comprising the non-rodent antigen of interest or a cell expressing the non-rodent antigen of interest.
 78. A method of screening drug candidate that targets an antigen of interest comprising a. introducing the antigen of interest into a genetically modified rodent as defined by any of claims 1-59, b. contacting said rodent with a drug candidate of interest, wherein the drug candidate is directed against the human C1q and the antigen of interest, and c. assaying if the drug candidate is efficacious in preventing, reducing, or eliminating cells characterized by the presence or expression of the antigen of interest.
 79. The method of claim 78, wherein the step of introducing comprises expressing in the rodent the antigen of interest.
 80. The method of claim 78, wherein the step of introducing comprises introducing into said rodent a cell expressing the antigen of interest.
 81. The method of any of claims 78-80, wherein the antigen of interest is a tumor associated antigen.
 82. The method of any of claims 78-80, wherein the cell is a bacterial cell such as a Staphylococcus cell, and the antigen is a bacterial antigen such as a Staphylococcus antigen.
 83. The method of any of claims 78-80, wherein the antigen is a viral antigen.
 84. The method of claim 82 or 83, wherein the step of introducing comprises infecting the rodent with the antigen of interest.
 85. The method of claim 78, wherein the rodent is an immunocompetent mouse or an immunocompetent rat.
 86. The method of claim 78, wherein the drug candidate is an antibody or an antigen-binding protein.
 87. The method of claim 86, wherein the antibody or the antigen-binding protein is a bispecific antibody or a bispecific antigen-binding protein, respectively, which is capable of binding both human C1q protein and the antigen of interest.
 88. A method for assessing whether a candidate antibody activates complement pathway, comprising a. providing a cell expressing an antigen of interest on the cell surface, a candidate antibody comprising a human Fc region and directed to the antigen of interest, and a serum sample from a genetically modified non-human animal of any of claims 1-59; b. mixing the cell with the candidate antibody to allow the antibody to bind to the antigen of interest expressed on the cell surface; c. adding the serum sample to the cell-antibody mixture to permit binding of the humanized C1q proteins in the serum sample to antibodies bound to the antigen of interest on the cell; and d. measuring cytotoxicity of the cell.
 89. A method for assessing a candidate bispecific antibody targeting an antigen of interest and human C1q, comprising a. mixing a cell or virus expressing the antigen of interest, the candidate bispecific antibody, and a blood sample from a genetically modified non-human animal of any of claims 1-59, and incubating to allow the antibody to bind to the antigen of interest expressed by the cell or virus and to the humanized C1q molecules in the blood sample; and b. measuring survival of the cell or virus.
 90. The method of claim 89, wherein the cell is a bacterial cell such as a Staphylococcus cell, and the antigen is a bacterial antigen such as a Staphylococcus antigen.
 91. The genetically modified non-human animal of claim 57, wherein the rat comprises in its genome: a. at the endogenous C1qa locus a nucleic acid sequence encoding a chimeric rat/human C1qa polypeptide which comprises the amino acid sequence of SEQ ID NO: 55; b. at the endogenous C1qb locus a nucleic acid sequence encoding a chimeric rat/human C1qb polypeptide which comprises the amino acid sequence of SEQ ID NO: 56; and c. at the endogenous C1qa locus a nucleic acid sequence encoding a chimeric rat/human C1qc polypeptide which comprises the amino acid sequence of SEQ ID NO:
 57. 92. The genetically modified non-human animal of claim 58, wherein the mouse comprises in its genome: a. at the endogenous C1qa locus a nucleic acid sequence encoding a chimeric mouse/human C1qa polypeptide which comprises the amino acid sequence of SEQ ID NO: 10; b. at the endogenous C1qb locus a nucleic acid sequence encoding a chimeric mouse/human C1qb polypeptide which comprises the amino acid sequence of SEQ ID NO: 11; and c. at the endogenous C1qa locus a nucleic acid sequence encoding a chimeric mouse/human C1qc polypeptide which comprises the amino acid sequence of SEQ ID NO:
 12. 